Protected fluorescent reagent compounds

ABSTRACT

Protected fluorescent reagent compounds and their methods of synthesis are provided. The compounds are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The compounds contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/966,334, filed on Apr. 30, 2018, which is a continuation of U.S.patent application Ser. No. 14/452,497, filed on Aug. 5, 2014, now U.S.Pat. No. 9,957,291, which claims the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application No. 61/862,502, filed on Aug. 5, 2013, thedisclosures of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

The use of fluorescent optical signals in analytical systems isextremely powerful due to the sensitivity and selectivity of the signaland the variety and adaptability of the chemistry. Furthermore, theability to simultaneously measure signals of different wavelengths hasfacilitated the development of assays in which multiple reactions can beobserved at the same time. For example, the use of four-colorfluorescent systems in nucleotide sequencing reactions facilitates thedetection of all four bases in a single reaction solution. Such methodshave been employed in the “real-time” detection of incorporation events,where the act of incorporation gives rise to a signaling event that canbe detected. In particularly elegant methods, labeling components arecoupled to portions of the nucleotides that are removed during theincorporation event, eliminating any need to remove such labelingcomponents before the next nucleotide is added. See, e.g., Eid, J. etal. (2009) Science 323:133-138.

At the same time, however, the exquisite sensitivity of fluorescentprobes, and the requirement that the probes be excited to potentiallyunstable electronic states in order for them to be detected, means thatthe fluorescent probes may be damaged during the course of the reactionor may inflict damage on other components of the reaction mixture. Suchdamage is particularly problematic in highly processive reactions, wherethe reaction mixture may be exposed to excitation radiation for extendedperiods of time. In enzyme-mediated template-dependent DNA sequencingmethods, such as fluorescent based single molecule, real time sequencingreactions, for example, the solution is exposed to excitation radiationwhile the sequencing reaction is occurring. If the enzyme or othercomponents of the reaction mixture are damaged due to such irradiation,the sequencing reaction can become compromised or end. For example, theenzyme may be inactivated due to interactions with excited dyes, whichare typically in close proximity to the enzyme during an incorporationevent.

There is therefore a continuing need to increase the performance offluorescence-based analytical systems. In particular, there is acontinuing need to develop fluorescent reagents that are readilydetectable at low concentrations and at convenient wavelengths, that areless sensitive to photodegradation than traditional fluorescentreagents, that are less likely to photodamage or otherwise compromiseother components of the analytical system, and that display otherdesirable characteristics.

SUMMARY OF THE INVENTION

The present disclosure addresses these and other needs by providing inone aspect protected fluorescent reagent compounds having structuralformula (I):Z—[S′—B′]_(m)  (I); wherein

Z is a multivalent central core element comprising a fluorescent dyeelement;

each S′ is independently an intermediate chemical group, wherein atleast one S′ comprises a shield element;

each B′ is independently a terminal chemical group, wherein at least oneB′ comprises a binding element; and

m is an integer from 2 to 24.

In some embodiments, the shield element decreases photodamage of thecompound or of a biomolecule associated with the binding element, and insome embodiments, the shield element decreases contact between thefluorescent dye and the binding element.

In specific embodiments, the shield element comprises a plurality ofside chains. In more specific embodiments, at least one side chain has amolecular weight of at least 300, and in even more specific embodiments,all of the side chains have a molecular weight of at least 300.

According to some embodiments, at least one side chain comprises adendrimer, a polyethylene glycol, or a negatively-charged component.According to specific embodiments, the negatively-charged componentcomprises a sulfonic acid.

In some embodiments, at least one side chain comprises a substitutedphenyl group, and in more specific embodiments, the at least one sidechain comprises the structure:

wherein each x is independently an integer from 1 to 6, or morespecifically an integer from 1 to 4. In some embodiments, at least oneside chain comprises a triazole, at least one side chain comprises thestructure:

or at least one side chain comprises the structure:

In some embodiments, the shield element comprises the structure:

wherein each y is independently an integer from 1 to 6, and in someembodiments the shield element comprises an inner layer and an outerlayer.

According to certain embodiments, at least one S′—B′ group comprises thestructure:

wherein R₁ and R₂ is each independently a side chain;L is an alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, orheteroaryl linker; and

In certain embodiments, the binding element comprises a nucleotide,biotin, or a polyphosphate.

According to certain embodiments, Z may comprise a branching element,and in more specific embodiments, the branching element may comprise asubstituted phenyl group or the branching element may comprise thestructure:

wherein each z′ is independently 1 to 6.

In certain compounds of formula (I), m is an integer from 2 to 12, morespecifically an integer from 2 to 8, and even more specifically aninteger from 2 to 4.

According to certain embodiments, Z may comprise a multivalentfluorescent dye element, and in specific embodiments, the multivalentfluorescent dye element may be a multivalent cyanine dye.

In certain specific embodiments, the multivalent cyanine dye has theformula:

wherein the A-ring and B-ring are independently monocyclic, bicyclic orpolycyclic aryl or heteroaryl moieties;Q is —(CH═C(R^(u)))_(c)—CH═), wherein c is 1, 2, 3, 4, or 5;each R^(w) and R^(z) is independently a substituted or unsubstitutedalkyl, heteroalkyl, aryl, or heteroaryl group that is coupled to theA-ring or B-ring either directly or through a carbonyl, amide,carbamide, ester, thioester, ether, thioether, or amino linkage;each R^(x) and R^(y), is independently an alkyl or heteroalkyl group,optionally substituted with a sulfonic acid, carboxylic acid, phosphonicacid, or phosphoric acid; andeach R^(u) is independently hydrogen, alkyl, or heteroalkyl.

In some embodiments, the compound of formula (I) has the structure:

wherein

n=1 or 2. In other embodiments, the compound has the structure:

In some embodiments, the Z group of formula (I) comprises a linker groupand, more specifically, the linker group may comprise a substitutedphenyl group. Even more specifically, the linker group may comprise thestructure:

wherein each z is independently an integer from 1 to 6 or even from 1 to4. In some embodiments, the linker group comprises a diaminoalkyl group.

According to another aspect of the invention, the disclosure providescompounds having structural formula (IIa) or (IIb):

wherein

X is a non-fluorescent multivalent central core element;

at least one D is a fluorescent dye element;

at least one W, if present, is a branching element;

n is an integer from 2 to 6;

each o is independently an integer from 1 to 4; and

each p is independently an integer from 1 to 4.

Also provided in another aspect of the invention are compounds havingstructural formula (III):

wherein

X is a non-fluorescent multivalent central core element;

at least one D is a fluorescent dye element;

at least one W is a branching element;

n is an integer from 2 to 6;

each p′ is independently an integer from 1 to 4; and

each p″ is independently an integer from 1 to 4.

In some embodiments of formulae (IIa), (IIb), and (III), the X groupcomprises a polyamine and, in more specific embodiments, the polyaminehas the structure:

In some embodiments, the X group comprises a substituted cyclohexane, ormore specifically a 1,3,5-triamino-cyclohexane.

In some embodiments, the X group comprises a substituted 1,3,5-triazine.

In some embodiments, the X group comprises a substituted benzene, ormore specifically the substituted benzene is

In some embodiments of these formulae, the compound comprises at leastone donor fluorescent dye element and at least one acceptor fluorescentdye element.

According to other embodiments, the shield element comprises a pluralityof side chains, including embodiments wherein at least one side chainhas a molecular weight of at least 300, wherein all of the side chainshave a molecular weight of at least 300, wherein the shield elementcomprises a dendrimer, and wherein at least one side chain comprises apolyethylene glycol. In some embodiments, at least one side chaincomprises a negatively-charged component, including embodiments whereinthe negatively-charged component comprises a sulfonic acid. In someembodiments, at least one side chain comprises a substituted phenylgroup, including embodiments wherein the at least one side chaincomprises the structure:

wherein each x is independently an integer from 1 to 6 or even from 1 to4.

In some embodiments, at least one side chain comprises a triazole, andin other embodiments, at least one side chain comprises the structure:

In specific embodiments, at least one side chain comprises thestructure:

According to some embodiments, the shield element comprises thestructure:

wherein each y is independently an integer from 1 to 6, and in someembodiments the shield element comprises an inner layer and an outerlayer.

In specific embodiments, at least one S′—B′ group comprises thestructure:

wherein R₁ and R₂ is each independently a side chain;L is an alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, orheteroaryl linker; and

In some embodiments, the binding element comprises a nucleotide, biotin,or a polyphosphate.

In certain embodiments, the branching element comprises a substitutedphenyl group, and in some embodiments the branching element comprisesthe structure:

wherein each z′ is independently an integer from 1 to 6, or even from 1to 4.

According to some embodiments of the above formulae, n may be an integerfrom 2 to 4, each o may be independently an integer from 1 to 3, each pmay be independently an integer from 1 to 3, each p′ may beindependently an integer from 1 to 3, or each p″ may be independently aninteger from 1 to 3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B schematically illustrate an exemplary nucleic acidsequencing process that can be carried out using aspects of theinvention.

FIG. 2A illustrates single molecule real time sequencing with aconventional nucleotide analog. FIG. 2B illustrates an exemplary nucleicacid sequencing process making use of a protected fluorescent reagent ofthe invention.

FIGS. 3A-3B display the structures and fluorescence spectra of twoexemplary protected fluorescent reagent compounds of the invention.

FIG. 4 illustrates the brightness of two exemplary protected fluorescentreagent compounds under sequencing conditions compared to control,unprotected compounds.

FIG. 5 shows a comparison of error types of two exemplary protectedfluorescent reagent compounds compared to control, unprotectedcompounds.

FIGS. 6A-6D show a comparison of sequencing readlength and accuracy fortwo exemplary protected fluorescent reagent compounds compared tocontrol, unprotected compounds.

FIG. 7 illustrates the brightness of two exemplary protected fluorescentreagent compounds under longer sequencing conditions compared tocontrol, unprotected compounds.

FIGS. 8A-8B illustrate a comparison of noise level variation duringincorporation reactions for the various reagent mixtures as measured intwo different sequencing channels under longer sequencing times.

FIGS. 9A-9C illustrate a decrease in the occurrence of photodamage forprotected compounds under longer sequencing times.

FIGS. 10A-10C demonstrate the effect of increased substitution of aprotected compound containing a triple-layer shield on photodamage.

FIG. 11 illustrates exemplary protected fluorescent compounds.

FIG. 12 illustrates non-limiting examples of azido-containing centralcore intermediate compounds.

FIG. 13 illustrates exemplary shield element-binding element (S′—B′)reagents.

FIG. 14 illustrates an exemplary synthetic route for preparing exemplaryshield core elements.

FIG. 15 illustrates an exemplary synthetic route for preparing exemplarymultivalent fluorescent dye core elements.

FIG. 16 illustrates an exemplary synthetic route for preparing exemplaryalternative core elements comprising fluorescent dye elements.

FIG. 17 illustrates exemplary core structures.

FIG. 18 illustrates an exemplary core element comprising fluorescent dyeelements.

FIG. 19 illustrates an exemplary synthetic route for preparing exemplaryintermediate fluorescent core elements.

FIG. 20 illustrates increased branching of fluorescent core elements.

FIG. 21 illustrates an exemplary synthetic route for the assembly of anexemplary protected compound.

FIGS. 22A-22H illustrate exemplary protected fluorescent reagentcompounds.

FIG. 23 illustrates an exemplary reaction showing the attachment ofalkyne-substituted shield element-binding elements (S′—B′s) to anazide-substituted dye core.

FIGS. 24-26 illustrate the synthesis of exemplary protected fluorescentreagent compounds.

FIG. 27 illustrates the assembly of exemplary compounds.

FIG. 28 illustrates the assembly of an exemplary protected fluorescentreagent compound.

FIG. 29 illustrates exemplary reactions useful in the attachment ofbinding elements to shield elements.

FIG. 30 illustrates the structure of N₃-Aba-Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P(5-10).

DETAILED DESCRIPTION OF THE INVENTION

Fluorescent reagents are used in a wide variety of differentapplications. Such applications may include the analysis of singlemolecules, and may involve observing, for example, single biomoleculesin real time as they carry out reactions. For ease of discussion, suchfluorescent reagents, and particularly the protected fluorescentreagents of the instant disclosure, are discussed herein in terms of apreferred application: the analysis of nucleic acid sequenceinformation, and particularly, single molecule nucleic acid sequenceanalysis. The instant protected fluorescent reagents will, however, findutility for other purposes in other applications, such as, for example,other fluorescence-based analytical techniques, where non-protectedfluorescent reagents are now routinely used.

In the preferred application, single molecule primer extension reactionsare monitored in real-time, to identify the continued incorporation ofnucleotides in the extension product to elucidate the underlyingtemplate sequence. In such single molecule real time (or SMRT™)sequencing, the process of incorporation of nucleotides in apolymerase-mediated template dependent primer extension reaction ismonitored as it occurs. In preferred aspects, the template/polymeraseprimer complex is provided, typically immobilized, within an opticallyconfined region, such as a zero mode waveguide (ZMW), or proximal to thesurface of a transparent substrate, optical waveguide, or the like (seee.g., U.S. Pat. Nos. 6,917,726, and 7,170,050 and U.S. PatentApplication Publication No. 2007/0134128, the full disclosures of whichare hereby incorporated by reference herein in their entirety for allpurposes). The optically confined region is illuminated with anappropriate excitation radiation for the fluorescently labelednucleotides that are to be used. Because the complex is within anoptically confined region, or very small illumination volume, only thereaction volume immediately surrounding the complex is subjected to theexcitation radiation. Accordingly, those fluorescently labelednucleotides that are interacting with the complex, e.g., during anincorporation event, are present within the illumination volume for asufficient time to identify them as having been incorporated.

A schematic illustration of this sequencing process is shown in FIGS. 1Aand 1B. As shown in FIG. 1A, an immobilized complex 102 of a polymeraseenzyme, a template nucleic acid and a primer sequence are providedwithin an observation volume (as shown by dashed line 104) of an opticalconfinement, of e.g., a zero mode waveguide 106. As an appropriatenucleotide analog, e.g., nucleotide 108, is incorporated into thenascent nucleic acid strand, it is illuminated for an extended period oftime corresponding to the retention time of the labeled nucleotideanalog within the observation volume during incorporation which producesa signal associated with that retention, e.g., signal pulse 112 as shownby the A trace in FIG. 1B. Once incorporated, the label that wasattached to the polyphosphate component of the labeled nucleotideanalog, is released. When the next appropriate nucleotide analog, e.g.,nucleotide 110, is contacted with the complex, it too is incorporated,giving rise to a corresponding signal 114 in the T trace of FIG. 1B. Bymonitoring the incorporation of bases into the nascent strand, asdictated by the underlying complementarity of the template sequence,long stretches of sequence information of the template can be obtained.

As just noted, in single-molecule real-time sequencing usingfluorescence detection, the polymerase enzyme is illuminated withexcitation light while a sequencing reaction is taking place. In somecases, this illumination results in photodamage which inhibits, and insome cases completely inactivates, the polymerase enzyme. This damagecan thus cause the sequencing reaction to end, resulting in shorter readlengths than desired. Significantly longer read lengths can in somecases be obtained in the dark than can be obtained for the samesequencing reaction under illumination. Importantly, damage to theenzyme under illumination may be accompanied by the formation of acovalent bond between a fluorescent dye moiety on a nucleotide analogand the polymerase enzyme and/or exchange of electrons between theexcited state dye and the enzyme. It is therefore believed that thestability of the enzyme can be compromised when there is contact betweenthe enzyme and a fluorescent moiety on a nucleotide analog which is inthe active site of the enzyme. In some cases, it appears that thismechanism constitutes the dominant mode of degradation. The instantdisclosure addresses the problem of photodamage by providing fluorescentcompounds that are less susceptible to such photodamage and that areless likely to cause photodamage to associated biomolecules, such asenzymes, that specifically bind to the fluorescent compounds.

U.S. patent application Ser. No. 13/767,619, filed Feb. 14, 2013, whichis incorporated by reference herein in its entirety for all purposes,provides a novel approach to mitigating photodamage and improvingsequencing readlengths by incorporation of a shielding protein into anucleotide analog. The nucleotide analog is constructed such that theshielding protein is disposed between the nucleotide phosphate portionand the fluorescent dye portion of the nucleotide analog. The size andposition of the protein are chosen such that the fluorescent dye portionof the analog does not come into contact with the polymerase enzyme whenthe nucleotide portion is held within the active site of the polymerase.Preventing contact between the fluorescent dye and the polymeraseprevents the formation of a covalent bond to the polymerase. Byshielding the enzyme from contact with the fluorescent dye, the proteinblocks a significant photodamage pathway, resulting in longer enzymelife under illumination.

The instant inventors have now discovered that photodamage can also bemitigated in fluorescent reagent compounds by the covalent incorporationof a general shield element between a fluorescent dye within thecompound and a binding element moiety. These compounds may also benefitfrom completely surrounding the fluorescent dye moiety by a protectiveshell of associated shield elements. Without intending to be bound bytheory, the shield element is thus understood to limit contacts betweenthe fluorescent core and the binding element and thus to minimizephotodamage, in particular photodamage to biomolecules that arespecifically associated with the binding element of the protectedcompound. The protected fluorescent reagents of the instant disclosurecan therefore be used in reactions where they are present, together withtheir associated biomolecular binding partners, for extended periods oftime, and where photodamage and the associated inactivation of theassociated biomolecules is therefore likely to be problematic. Theimproved performance of the instant fluorescent reagent compoundsprovides advantages for the use of these reagents in various fluorescentanalytical techniques.

FIGS. 2A and 2B provide a schematic illustration of the role believed tobe played by the shield element of the instant compounds in the contextof a single-molecule real-time sequencing reaction. FIG. 2A illustratesreal-time single-molecule sequencing with a conventional nucleotideanalog. The polymerase enzyme 210 is bound to the surface of asubstrate, such as a chip, 260, for example with a linker 250. Thepolymerase enzyme 210 is complexed with a template nucleic acid having atemplate strand 220 and a primer/growing strand 230. Sequencing isperformed by observing the enzyme while it incorporates nucleotides intothe growing strand. A nucleotide analog being incorporated into thegrowing strand generally spends more time in the active site than anucleotide not being incorporated, allowing for the identification ofthe incorporated nucleotide by its fluorescent signal. When thenucleotide is incorporated, the remainder of the nucleotide analog,including the fluorescent label, is cleaved away and the enzyme is readyfor the next incorporation. By watching the incorporation of bases overtime, the sequence of the template nucleic acid 220 can be determined bydetermining the series of nucleotides that are incorporated into thegrowing strand 230. Variations in the general technique, such asattaching the polymerase to other surfaces or even other media, and/orrestricting movement of the polymerase complex in other ways, or even inusing other types of restricted volumes, may also be contemplated inthis general approach.

As just described, FIG. 2A (1) shows a conventional nucleotide analog240, held in the enzyme prior to incorporation. The nucleotide portionof the analog 244 is held in the active site of the enzyme in positionfor incorporation. FIG. 2A (2) illustrates that while the nucleotideanalog is associated with the enzyme, the fluorescent moiety 242 cancome into contact with the enzyme. During this process, the enzyme isilluminated with excitation light to allow for observation of thefluorescent moiety. When the fluorescent moiety absorbs the excitationlight, it enters into an excited state. It is believed that having theexcited fluorescent moiety in the vicinity of the enzyme, and inparticular when the fluorescent moiety comes into contact with theenzyme, damage to the enzyme may result, adversely affecting thesequencing reaction. The damage to the enzyme may result in slowing theenzyme, altering its effectiveness, or in completely halting thepolymerase reaction.

FIG. 2B shows a protected fluorescent compound of the invention (270)having three fluorescent dyes (272), three nucleotide binding elements(274), and three shield elements (276). The compound is thus trivalentwith respect to dye, trivalent with respect to binding elements, andhexavalent overall, as will be further described below. As would beunderstood by one of ordinary skill in the art, sequencing may becarried out with the instant protected reagent compounds by the sameprocesses described above for conventional nucleotide analogs. As with aconventional enzyme substrate, the nucleotide portion of the protectedreagent is held in the active site of the enzyme prior to incorporation.The protected reagent thus has a nucleotide binding element, 274,connected to the shield element through the phosphate portion of thenucleotide. In some embodiments, the nucleotide may be attached to theshield element through a linker group. Without intending to be bound bytheory, the shielding elements are thought to prevent contact betweenthe polymerase enzyme and the fluorescent dye moiety, 272. By shieldingthe enzyme from contact with the fluorescent dye moiety while thenucleotide binding element portion of the analog is in the active siteof the enzyme, the enzyme is protected from photodamage due to contactwith the fluorescent moiety's excited state. Although the sequencingreaction is exemplified here with a particular embodiment of the instantcompounds, it should be understood that any other suitable compoundembodiment could be utilized in such reactions and would be consideredwithin the scope of the invention.

While the usefulness of the protected reagents of the invention isillustrated with the description above of SMRT™ sequencing, it is to beunderstood that these compounds may be used with any appropriateenzymatic or binding reaction and will thus have broader application inother analytical techniques. For example, the protected fluorescentreagents of the instant disclosure are also useful in the measurement ofany type of binding interaction, not just binding interactions thatresult in the reaction of the reagent. While in preferred embodiments,such as single-molecule, real-time nucleic acid sequencing reactions,the reagent serves as an enzyme substrate and is chemically altered as aresult of the interaction, in other embodiments, such as, for example,the binding of a protected fluorescent reagent to an antibody, areceptor, or other affinity agent, the reagent remains unaltered as aresult of the interaction. Measurement of the binding interaction, orany other type of interaction, may be performed using well-knownfluorescence techniques and biochemical processes. Examples of suchtechniques and processes include fluorescence resonance energy transfer(FRET), fluorescence cross-correlation spectroscopy, fluorescencequenching, fluorescence polarization, flow cytometry, and the like.

The instant disclosure provides chemical formulae and specific chemicalstructures for the inventive protected fluorescent reagents. Wherechemical moieties are specified by their conventional chemical formulae,written from left to right, they optionally equally encompass the moietywhich would result from writing the structure from right to left, e.g.,—CH₂O— is intended to also recite —OCH₂—; —NHS(O)₂— is also intended tooptionally represent. —S(O)₂HN—, etc. Moreover, where compounds can berepresented as free acids or free bases or salts thereof, therepresentation of a particular form, e.g., carboxylic or sulfonic acid,also discloses the other form, e.g., the deprotonated salt form, e.g.,the carboxylate or sulfonate salt. Appropriate counterions for salts arewell-known in the art, and the choice of a particular counterion for asalt of the invention is well within the abilities of those of skill inthe art. Similarly, where the salt is disclosed, this structure alsodiscloses the compound in a free acid or free base form. Methods ofmaking salts and free acids and free bases are well-known in the art.

“Cyanine,” as used herein, refers to polymethine dyes such as thosebased upon the cyanine, merocyanine, styryl and oxonol ring. Cyaninedyes include, for example, CY3, CY3.5, CY5 and CY5.5 type dyes.

As used herein, “nucleic acid” means any natural or non-naturalnucleotide or nucleoside phosphate oligomer or polymer; e.g., DNA, RNA,single-stranded, double-stranded, triple-stranded or more highlyaggregated hybridization motifs, and any chemical modifications thereof.

Exemplary modified nucleic acids include, but are not limited to,peptide nucleic acids (PNAs), those with phosphodiester groupmodifications (e.g., replacement of O⁻ with OR, NR, or SR), 2′-, 3′- and5′-position sugar modifications, modifications to the base moiety, e.g.,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications, i.e.,substitution of P(O)O₃ with another moiety, methylations, unusualbase-pairing combinations such as the isobases, isocytidine andisoguanidine and the like. Nucleic acids can also include non-naturalbases, e.g., nitroindole. Non-natural bases include bases that aremodified with a minor groove binder, an intercalating agent, ahybridization enhancer, a chelating agent, a metal chelate, a quencher,a fluorophore, a fluorogenic compound, etc. Modifications within thescope of “nucleic acid” also include 3′ and 5′ modifications with one ormore of the species described above.

Nucleic acids, nucleotides and nucleosides contain nucleobases. Inaddition to the naturally occurring nucleobases of deoxyribonucleicacids, i.e., adenine, cytosine, guanine, and thymine, the compounds ofthe invention may optionally include modified bases. These componentsmay also include modified sugars. For example, the nucleic acids,nucleotides, or nucleosides described herein may comprise at least onemodified base moiety which is selected from the group including, but notlimited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-d-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, -methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,nitroindole, and 2,6-diaminopurine.

Typically the nucleic acids, nucleotides, and nucleosides describedherein may comprise either ribose (RNA) or deoxyribose (DNA). In someembodiments, the nucleic acids, nucleotides, or nucleosides may comprisea modified sugar moiety selected from the group including, but notlimited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. Amodified sugar moiety may, for example, be present in a binding elementof the instant protected compounds.

In yet other embodiments, the nucleic acids, nucleotides, andnucleosides may comprise at least one modified phosphate backboneselected from the group including, but not limited to, a peptide nucleicacid hybrid, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof. A modified phosphate backbone may, for example, be present in abinding element of the instant protected compounds.

Nucleic acids, nucleotides, and nucleotides described herein may alsoinclude species that are modified at one or more internucleotide bridges(e.g., P(O)O₃) by replacing or derivatizing an oxygen of the bridgeatom. For example a “nucleic acid” also refers to species in which theP(O)O₂ moiety (the O⁻ moiety remains unchanged or is converted to “OR”)of a natural nucleic acid is replaced with a non-natural linker species,e.g., —ORP(O)O—, —ROP(O)R—, —ORP(O)OR—, —ROP(O)OR—, or —RP(O)R— in whichthe symbol “—” indicates the position of attachment of the linker to the2′-, 3′- or 5′-carbon of a nucleotide sugar moiety, thus allowing theplacement of the exemplified, and other, non-natural linkers betweenadjacent nucleoside sugar moieties. Exemplary linker subunits (“R”)include substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl moieties. Such modifications may be present ina binding element of the instant protected compounds

Further exemplary nucleic acids, nucleotides, and nucleosides describedherein may include a polyphosphate moiety, e.g., pyrophosphate or ahigher homologue, such as the 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-merand the like. The polyphosphate moieties of the instant protectedreagent compounds generally comprise from 2 to 10 phosphates. Inpreferred embodiments, the polyphosphate moieties comprise 4, 5, 6, 7 or8 phosphates. In other embodiments, a methylene moiety, NH moiety, or Smoiety bridges two or more phosphorus atoms, replacing the OPO link withan PCH₂P, PNHP, or PSP link.

Furthermore, compounds of the disclosure may include species in whichone or more internucleotide bridge does not include phosphorus. Anexemplary bridge includes a substituted or unsubstituted alkyl orsubstituted or unsubstituted heteroalkyl moiety in which a carbon atomis the locus for the interconnection of two nucleoside sugar residues.The discussion above is not limited to moieties that include a carbonatom as the point of attachment; the locus may also be anotherappropriate linking atom, such as nitrogen or another atom.

Phosphodiester linked nucleic acids described herein may be synthesizedby standard methods known in the art, e.g. by use of an automated DNAsynthesizer using commercially available amidite chemistries (Ozaki etal., Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al.,Nucleic Acids Research, 18: 5419-5423 (1990); Beaucage et al.,Tetrahedron, 48: 2223-2311 (1992); Molko et al., U.S. Pat. No.4,980,460; Koster et al., U.S. Pat. No. 4,725,677; Caruthers et al.,U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679). Nucleic acidsbearing modified phosphodiester linking groups may be synthesized bymethods known in the art. For example, phosphorothioate nucleic acidsmay be synthesized by the method of Stein et al. (Nucl. Acids Res.16:3209 (1988)), methylphosphonate nucleic acids may be prepared by useof controlled pore glass polymer supports (Sarin et al., Proc. Natl.Acad. Sci. U.S.A. 85:7448-7451 (1988)). Other methods of synthesizingboth phosphodiester- and modified phosphodiester-linked nucleic acidswill be apparent to those of skill in the art.

The nucleotides and nucleoside phosphates of the instant protectedreagent compounds are generally meant to be used as substrates forpolymerase enzymes, particularly in the context of nucleic acidsequencing. Therefore, generally, any non-natural base, sugar, orphosphate of the nucleotide or nucleoside phosphate may be included as anucleotide or nucleoside phosphate of the invention if the nucleosidephosphate is capable of acting as a substrate for any natural ormodified polymerase enzyme.

“Activated derivatives of carboxyl moieties,” and equivalent species,refers to a moiety on a component of the instant protected reagentcompounds or their precursors or derivatives or on another reagentcomponent in which an oxygen-containing, or other, leaving group isformally accessed through a carboxyl moiety, e.g., an active ester, acylhalide, acyl imidazolide, etc. Such activated moieties may be useful incoupling the various components of the instant compounds as they areassembled.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturated alkylradicals include, but are not limited to, groups such as methyl,methylene, ethyl, ethylene, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl, and the like. An unsaturated alkyl group is one having one ormore double bonds or triple bonds. Examples of unsaturated alkyl groupsinclude, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, includes “alkylene,”“alkynyl” and, optionally, those derivatives of alkyl defined in moredetail below, such as “heteroalkyl”.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, S, P and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene, alkynyl,and heteroalkylene linking groups, no orientation of the linking groupis implied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Also included aredi- and multi-valent species such as “cycloalkylene.” Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to, speciessuch as trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings), which are fused togetheror linked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from one to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. A heteroaryl group canbe attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Also included are di- and multi-valentlinker species, such as “arylene.” Substituents for each of the abovenoted aryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Exemplary substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, SO₃R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. Accordingly,from the above discussion of substituents, one of skill in the art willunderstand that the terms “substituted alkyl” and “heteroalkyl” aremeant to include groups that have carbon atoms bound to groups otherthan hydrogen atoms, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl(e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The substituents set forth in the paragraph above are referred to hereinas “alkyl group substituents.”

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″)═NR″,—S(O)R′, —S(O)₂R′, SO₃R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃,—CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a numberranging from zero to the total number of open valences on the aromaticring system; and where R′, R″, R′″ and R″″ are preferably independentlyselected from hydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryland heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl. When a compound of the invention includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R′″ and R″″ groups when more than one ofthese groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)-J-(CR″R″)_(d)—, where s and d are independently integers offrom 0 to 3, and J is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)/NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)-alkyl.

The substituents set forth in the two paragraphs above are referred toherein as “aryl group substituents.”

When referring to components of the compounds of the invention, the term“residue derived from,” refers to a residue formed by the reaction of afirst reactive functional group on a first component (e.g., central coreelement, dye element, shield element, or a binding element) and a secondreactive functional group on a second component (e.g., central coreelement, dye element, shield element, or a binding element) to form acovalent bond. In exemplary embodiments, an amine group on the firstcomponent is reacted with an activated carboxyl group on the secondcomponent to form a residue including one or more amide moieties. Otherpermutations of first and second reactive functional groups areencompassed by the invention. For example, the copper-catalyzed reactionof an azide-substituted first component with an alkyne-substitutedsecond component results in a triazole-containing residue through thewell-known “click” reaction, as would be understood by those of ordinaryskill in the art. See Kolb et al. (2001) Angew. Chem. Int. Ed. Engl.40:2004; Evans (2007) Aus. J. Chem. 60:384.

In some embodiments, a copper-free variant of the click reaction may beused to couple the first and second reactive groups. See, e.g., Baskinet al. (2007) Proc. Natl Acad. Sci. U.S.A. 104:16793-97. For example, anazide-substituted first component may be reacted with a cycloalkyne,ideally a cyclooctyne, attached to the second component, in the absenceof a copper catalyst. Such so-called copper-free click reagents areavailable commercially. Examples of such cycloalkynes include, withoutlimitation, dibenzocyclooctyne-amine, bicyclo[6.1.0]non-4-yn-9-yl, ormonofluorinated cyclooctynes. Other coupling chemistries may also beusefully employed in the synthesis of the compounds of the instantdisclosure, as would be understood by those of skill in the art.

It should also be understood that the attachment sites for the first andsecond reactive functional groups in the just-described reactions cangenerally be reversed if so desired, depending on the situation. Forexample, in the case of a “click” reaction, the first component may beazide-substituted and the second component may be alkyne-substituted, asdescribed above, or the first component may be alkyne-substituted andthe second component may be azide-substituted. Such variation in thereactions is well within the skill of those in the art.

As used herein, a listed range of integers includes each of the integerswithin the range. For example, an integer from 2 to 6 includes theintegers 2, 3, 4, 5, and 6.

Protected Fluorescent Reagent Compounds

The instant disclosure provides fluorescent reagent compounds for use inthe measurement and analysis of enzymatic reactions and other molecularrecognition events, such as, for example, the single-molecule real-timesequencing of nucleic acids, the binding of fluorescent compounds tobiomolecules such as receptors and antibodies, and other types ofreaction and recognition events, where the target biomolecules areprotected from photodamage or other detrimental reactions by thefluorescent compounds due to the specific structural design of thecompounds. The protection may also extend to protection from photodamageor other detrimental reactions that may occur to the reagent compoundsthemselves.

Accordingly, in one aspect, the disclosure provides compounds ofstructural formula (I):Z—[S′—B′]_(m)  (I); wherein

Z is a multivalent central core element comprising a fluorescent dyeelement;

S′ is an intermediate chemical group, wherein at least one S′ comprisesa shield element;

B′ is a terminal chemical group, wherein at least one B′ comprises abinding element; and

m is an integer from 2 to 24.

As described in more detail elsewhere in the disclosure, it is believedthat the shield element of the instant compounds protects againstphotodamage of biomolecules associated with the binding element of thecompounds. In particular, and without intending to be bound by theory,biomolecules capable of recognizing and binding to the compounds of thedisclosure, for example receptors, antibodies, enzymes, and the like,may be protected from photodamage due to steric or other effects,whereby the large shield elements within the instant compounds decreasecontact between the fluorescent dyes within the compounds and abiomolecule associated with the binding element. The shield elements mayalternatively, or in addition, provide a protective microenvironment forthe attached fluorescent dyes that results in improved physical andchemical properties of the dyes.

It should be understood throughout the disclosure, that an intermediatechemical group, S′, of the instant compounds, while potentiallycorresponding to any suitable intermediate chemical group, preferablycomprises a shield element. It should further be understood that aterminal chemical group, B′, of the instant compounds, while potentiallycorresponding to any suitable terminal chemical group, preferablycomprises a binding element. This does not exclude the possibility,however, that in some embodiments, one or more intermediate chemicalgroups will not comprise a shield element or that one or more terminalchemical groups will not comprise a binding element, in any possiblecombination. It should also be understood that within a given protectedreagent compound of the instant disclosure, the S′ groups may be thesame or different from one another, depending on the desired properties,the B′ groups may be the same or different from one another, likewisedepending on the desired properties, and the variation in S′ and B′ mayoccur in any possible combination. In preferred compound embodiments ofthe instant formulae, however, all S′ groups are the same, and all B′groups are the same. In highly preferred compound embodiments of theinstant formulae, all S′ groups are the same, and they all comprise ashield element, and all B′ groups are the same, and they all comprise abinding element.

Multivalent Central Core Elements

One of the advantages of the protected fluorescent compounds of theinstant disclosure is the multivalency of binding elements within eachcompound molecule. Specifically, each compound molecule comprises aplurality of terminal chemical groups, wherein at least one of theterminal chemical groups comprises a binding element that is capable ofbeing recognized and bound by a target of interest. In the case ofenzyme targets, such as, for example, the DNA polymerase used insingle-molecule real-time DNA sequencing assays, the binding elementcomprises a substrate component capable of being reacted upon by theenzyme. A protected reagent compound for use in a DNApolymerase-catalyzed reaction might therefore include a nucleobase suchas adenine, cytosine, guanine, thymine, or a modified form of one ofthese bases. In the normal DNA polymerase reaction using natural NTPs,or specifically dNTPs, the bond between the alpha phosphate, attached tothe nucleoside and the beta phosphate is cleaved, releasingpyrophosphate, whereas in the instant reagent compounds, turnover by theenzyme releases the rest of the protected compound from the nucleobase.Multivalency of such binding elements on the protected fluorescentreagent therefore allows the same reagent molecule to undergo multipleenzyme turnovers before the molecule loses reactivity. The multivalencythus effectively increases the concentration of enzyme substrate in thereaction. Multivalency of binding elements in the protected reagentcompounds is likely to provide similar advantages in the analysis ofother types of binding interactions as well.

The integer “m” in formula (I) indicates the multivalency of S′—B′groups in the compounds of this formula. In some embodiments, m is aninteger from 2 to 24. In specific embodiments, m is an integer from 2 to12. In more specific embodiments, m is an integer from 2 to 8 or from 2to 4. It should be understood that in all of these embodiments, thevalue of m can include any of the integer values within the listedrange.

The multivalency of binding elements in the instant protectedfluorescent compounds results, at least in part, from the inclusion of amultivalent central core element within the reagent molecule. Accordingto one aspect, the multivalent central core element of the instantfluorescent compounds comprises a multivalent fluorescent dye element.Any suitable fluorescent dye may be used in such a fluorescentmultivalent central core element, Z, provided the dye contains aplurality of reactive sites suitable for attachment of the relevantintermediate chemical groups, S′, and terminal chemical groups, B′. Inpreferred embodiments, the fluorescent dye is a cyanine dye, for exampleany of the cyanine dyes disclosed in co-owned PCT InternationalPublication No. 2012/027618; U.S. Patent Application Publication No.2012/0058469; U.S. Patent Application Publication No. 2012/0058482; U.S.Patent Application Publication No. 2012/0052506; and U.S. PatentApplication No. 61/649,058, the disclosures of each of which areincorporated herein by reference in their entireties for all purposes.

As noted in the above references, exemplary cyanine dyes have theformula:

wherein the A-ring and B-ring are independently selected frommonocyclic, bicyclic or polycyclic aryl or heteroaryl moieties. Q is asubstituted or unsubstituted methine moiety (e.g.,—(CH═C(R^(u)))_(c)—CH═), in which c is an integer selected from 1, 2, 3,4, or 5. Each R^(u), R^(w), R^(x), R^(y) and R^(z) is independentlyselected from various suitable substituents, and the indices w and z areindependently selected from the integers from 0 to 6. For use in afluorescent multivalent central core element, Z, of the instantcompounds, at least two of the R^(w) and/or R^(z) groups would beunderstood to contain suitable functionality to allow for the attachmentof a plurality of S′—B′ groups.

In some embodiments, each R^(w) and R^(z) is independently a substitutedor unsubstituted alkyl, heteroalkyl, aryl, or heteroaryl group that iscoupled to the A-ring or B-ring either directly or through a carbonyl,amide, carbamide, ester, thioester, ether, thioether, or amino linkage.

In some embodiments, each R^(x) and R^(y), is independently an alkyl orheteroalkyl group, optionally substituted with a sulfonic acid,carboxylic acid, phosphonic acid, or phosphoric acid.

In some embodiments, each R^(u) is independently hydrogen, alkyl, orheteroalkyl.

Specific embodiments are described more thoroughly in the above-listedpatent publications.

A specific example of a protected fluorescent reagent compounds of theinstant disclosure comprising a tetravalent cyanine dye is the followingstructure:

where n is 1 or 2. It should be understood that the connections betweenthe cyanine dye and the S′—B′ groups, in this, and in other relatedcompounds, may be varied as desired. Such variation depends on the exactlinking reactions used to attach the S′—B′ group, as would be understoodby those of ordinary skill in the art. Exemplary linkage reactions aredescribed further below and are also described in disclosures of theabove-listed, co-owned patent application publications.

Other examples of protected reagent compounds comprising multivalentcyanine dyes, including tetravalent, hexavalent, octavalent, anddodecavalent cyanine dyes, include the following non-limiting compoundstructures:

The above compound examples are labeled with the name of the specificdye that serves as the building block for the multivalent central coreelement and the name of the linking moiety that provides branchingfunctionality. The S′—B′ valency is indicated in parentheses for eachcompound. It should be understood that the linking moieties, and thefunctional groups used to couple them both to the central dye moleculeand to the S′—B′ groups may be varied as desired within the scope of theinstant disclosure.

As is readily apparent upon review of the above structures, the S′—B′groups may be attached directly, or nearly directly, to the dyemolecules, or they may be attached indirectly, for example, through alinker group. In some embodiments, the linker group comprises thestructure:

wherein each z is independently an integer from 1 to 6. In more specificembodiments, each z is independently an integer from 1 to 4. As isapparent in some of the above-described compound examples, the linkergroup may further comprise an aminoalkyl group or a diaminoalkyl group.Other linker groups, for example, acylalkyl groups, diacylalkyl groups,or any other suitable linker group, may be usefully employed in theattachment of the S′—B′ groups to the fluorescent multivalent centralcore element, as would be understood by those of ordinary skill in theart.

The linking moieties may also provide branching functionality, as isdescribed more thoroughly below in the section entitled “BranchingElements”, thus increasing the total possible valency of the fluorescentmultivalent central core element.

Non-Fluorescent Multivalent Central Core Elements

According to another aspect, the instant compounds comprise amultivalent central core element that is not itself fluorescent but thatrather provides sites of attachment for a plurality of fluorescent dyeelements to the core element. The fluorescent dye elements themselvesmay be either monovalent or multivalent, depending on the intended useand desired properties. The non-fluorescent multivalent core elementthus serves as a “scaffold” for assembly of the fluorescent dye elementsand binding elements into the larger molecule. Exemplary scaffoldsusefully employed in the protected fluorescent compounds of the instantdisclosure are described in co-owned U.S. Patent Application PublicationNo. 2012/0077189, which is incorporated herein by reference in itsentirety for all purposes.

In preferred embodiments, the non-fluorescent multivalent central coreelement provides for the attachment of a plurality of fluorescent dyesto the core. For example, the disclosure provides compounds ofstructural formula (IIa) or (IIb):

wherein

-   -   X is a non-fluorescent multivalent central core element;    -   at least one D is a fluorescent dye element;    -   at least one W, if present, is a branching element;    -   n is an integer from 2 to 6;    -   each o is independently an integer from 1 to 4;    -   each p is independently an integer from 1 to 4; and S′ and B′        are as defined above.

As is apparent from the above formulae, the non-fluorescent multivalentcore element, X, is attached to a plurality of D groups, at least one ofwhich is a fluorescent dye. The D groups of formulae (IIa) and (IIb) areaccordingly either bivalent (e.g., in formula (IIa), and also in formula(IIb), when o=1) or have higher valency (e.g., in formula (IIb), wheno≥2). The D groups thus serve as a link between the X group and the Wgroup or groups (for formula (IIb)) or between the X group and the S′—B′group or groups (for formula (IIa).

For D groups that are fluorescent dye elements, the fluorescent dyeelements are therefore either bivalent or have higher valency. Anysuitable fluorescent dye may be used in the instant protected compounds,provided that the dye has proper valency and can be appropriatelycoupled to the relevant associated groups (X and W or S′). In preferredembodiments, the fluorescent dye is a cyanine dye, as described indetail above, including the cyanine dyes disclosed in the above-citedpatent publications and in the above-depicted structures.

In other preferred embodiments, the D groups are monovalent and do notprovide a connection between the non-fluorescent multivalent centralcore element and the S′—B′ groups but are instead terminal groups thatare connected to the central core element through a branching elementthat also serves to link the S′—B′ groups to the central core element.For example, the disclosure provides protected reagent compounds ofstructural formula (III):

wherein

-   -   X is a non-fluorescent multivalent central core element;    -   at least one D is a fluorescent dye element;    -   at least one W is a branching element;    -   n is an integer from 1 to 6;    -   each p′ is independently an integer from 1 to 4;    -   each p″ is independently an integer from 1 to 4;        and S′ and B′ are as defined above.

As is evident in formula (III), the D group or groups and the S′—B′group or groups are attached to the non-fluorescent multivalent centralcore element through an intermediary branching element, W. Suchbranching elements have already been mentioned above in the context ofthe linker groups of the protected reagent compounds of formula (I), andthey will described more thoroughly below in the section entitled“Branching Elements”.

In preferred embodiments of the compounds of formulae (IIa), (IIb), and(III), all S′ groups are the same and all B′ groups are the same withina given reagent compound. In highly preferred embodiments, for a givenreagent compound, all S′ groups are the same, and each S′ groupcomprises a shield element, and all B′ groups are the same, and each B′group comprises a binding element.

As is evident in the structures of the compounds according to formulae(IIa), (IIb), and (III), the intermediate chemical group, S′, and itsassociated terminal chemical group, B′, may be either directly attachedto the non-fluorescent multivalent central core element or may beattached indirectly through either an intermediary multivalent dye orthrough a branching element. The specific chemical linkages used in anyof these attachments is not critical, so long as the linkages are stableunder the conditions in which the compounds are being used, as would beunderstood by those of ordinary skill in the art.

As mentioned above, the non-fluorescent multivalent central core elementaccording to this aspect of the invention serves as a scaffold for dyes,intermediate chemical groups and their associated shield elements, andterminal chemical groups and their associated binding elements in someof the disclosed compounds. In some embodiments, the non-fluorescentmultivalent central core element comprises a polyamine central coreelement. Polyamines may be readily reacted with appropriateelectrophilic reagents, such as electrophilic linker or branchingelements, dye reagents, and the like, to generate intermediate compoundsthat may in turn be reacted with appropriate shield element and bindingelement reagents. It should be understood that the order of suchreactions may be varied, depending on the desired outcome, as would beunderstood by those of ordinary skill in the art. Non-limiting examplesof polyamines usefully employed in the non-fluorescent multivalentcentral core elements of the instant disclosure include the following:

The skilled artisan would understand, however, that other polyaminescould be readily utilized in the protected fluorescent compounds of theinstant disclosure.

In specific embodiments, the non-fluorescent multivalent central coreelement comprises a substituted cyclohexane, more specifically a1,3,5-triamino-cyclohexane.

In other specific embodiments, the non-fluorescent multivalent centralcore element comprises a substituted 1,3,5-triazine.

In still other specific embodiments, the non-fluorescent multivalentcentral core element comprises a substituted benzene.

In some embodiments the non-fluorescent multivalent central core elementcomprises an ether linkage. In some embodiments, the non-fluorescentmultivalent central core element comprises an acyl linkage. Examples ofsuch ether and acyl-linked central core elements include the followingstructures:

These structures may be incorporated into the instant protected reagentcompounds as described in detail below. In particular, ether-linkedcentral core elements may be modified with acetylene-containing groups,including cycloalkyne-containing groups, and the acetylene groups maythen be coupled to azide-containing reagents using “click” chemistry or“copper-free click” chemistry. Likewise, carboxylate-containing centralcore elements may be activated using suitable reagents, and theactivated acyl groups may then be coupled to appropriate nucleophilicreagents as desired. Alternatively, or in addition, the central coreelements may be activated using azide-containing groups, and thosegroups may be coupled to acetylene-containing reagents, includingcycloalkyne-containing reagents, using “click” chemistry or “copper-freeclick” chemistry. Such reactions are well understood by those ofordinary skill in the art.Branching Elements

As described above, the protected reagent compounds of the instantdisclosure may, in some embodiments, comprise additional linker groupsthat may be attached, for example, to a central core element or that mayserve as an intervening linkage between a D group and S′—B′ group. Suchlinker groups may in some embodiments serve as branching elements toallow the attachment of multiple S′—B′ groups to D groups (as in Formula(IIb)) or multiple D and S′—B′ groups to X groups (as in Formula (III)).In preferred embodiments, the branching elements may include acyl groupsfor coupling to, for example, an appropriate amino group, amino groupsfor coupling to, for example, an appropriate acyl group, and furtherreactive groups, such as, for example, acetylene groups for coupling to,for example, azide-labeled groups. Exemplary branching elements usefullyemployed in the protected reagent compounds of the instant disclosureinclude the following:

wherein each z′ is independently an integer from 1 to 6. In morespecific embodiments, each z′ is independently an integer from 1 to 4.In some embodiments, the branching element includes the following:

In other specific embodiments, the branching element comprises thestructure:

and in some embodiments comprises the structure

In some embodiments, the branching element comprises the structure

Some branching elements may contain more than one of the abovestructures, and different branching element structures may be presentwithin a single molecule of the instant compounds.

As would be understood by those of ordinary skill in the art, theterminal acyl groups of the above structures are preferably linked to anamine group within a given compound, for example to an amine group inthe central core element, and the terminal amino groups of the abovestructures are preferably linked to an acyl group within a givencompound, for example to an acyl group in the D group (for compounds offormula (III)). Branching elements are typically coupled to theirassociated shield element-binding element (S′—B′) groups using “click”chemistry, or “copper-free click” chemistry, as described morethoroughly below. The terminal methylene groups in the above structuresare therefore preferably linked to the S′—B′ groups through a triazolestructure, although other linking chemistry should be considered withinthe scope of the invention.

It should be generally understood that other coupling chemistry mayprove suitable in the protected compounds of the instant invention, andthat the branching element structures should not, therefore, be limitedto those illustrated in the exemplified compounds. Accordingly,reactions other than those exemplified in the synthetic schemes below,may be suitable for generating the protected reagent compounds of theinstant invention. For example, alkylations, e.g., through the reactionof alkyl halides, acylations, and other suitable reactions may beutilized in synthesizing the instant compounds.

Intermediate Compounds

Exemplary intermediate compounds useful in the synthesis of someembodiments of the instant compounds comprise a non-fluorescentmultivalent central core element with attached fluorescent dye groups.These compounds further comprise acetylene groups that may beefficiently coupled to shield elements and their associated bindingelements through “click” reactions, or “copper-free click” reactions,and the like. Such synthetic intermediates include the followingnon-limiting examples:

The “dye” groups of the previous exemplary intermediates may be the sameor different fluorescent dyes within a given reagent molecule. Inpreferred embodiments, the dyes are different, and at least one of thedyes is an energy transfer “donor” dye and at least one of the dyes isan energy transfer “acceptor” dye. Compounds comprising donor andacceptor fluorophores thus provide for the possibility of measuring afluorescent signal using fluorescence resonance energy transfer (FRET)techniques or the like, as would be understood by those of ordinaryskill in the art. Examples of FRET-labeled nucleotides anddonor-acceptor pairing are provided in U.S. Patent ApplicationPublication Nos. 2010/0255488 and 2012/0058469, the full disclosures ofwhich are hereby incorporated by reference herein in their entirety forall purposes. Additional long-wavelength heteroarylcyanine dyes usefullyincorporated into the instant protected fluorescent compounds aredisclosed in U.S. patent application Ser. No. 13/898,369, filed May 20,2013, the full disclosure of which is hereby incorporated by referenceherein for all purposes.

The terms “fluorescence resonance energy transfer” and “FRET” are usedherein to refer to both radiative and non-radiative energy transferprocesses. For example, processes in which a photon is emitted and thoseinvolving long-range electron transfer are included within these terms.Throughout this specification, both of these phenomena are subsumedunder the general term “donor-acceptor energy transfer”.

Any of the dyes set forth herein can be a component of a FRET pair aseither the donor or acceptor. Conjugating a donor and an acceptorthrough reactive functional groups on the donor, the acceptor, and, ifappropriate, an appropriate linker or carrier molecule, is well withinthe abilities of those of skill in the art in view of the instantdisclosure.

Exemplary protected fluorescent compounds having one or more donor andone or more acceptor fluorophores include the following non-limitingstructures:

where the donor dye and acceptor dye attachment locations are asindicated, the R″ groups represent locations for attachment of the S′—B′groups, and where the lower two structures comprise alternativepolyamine central core elements.

Further examples of protected fluorescent compounds according to thisaspect of the instant invention include the nonlimiting structuresillustrated in FIG. 11,

where the R″ groups represent locations for attachment of the S′—B′groups, and where the structures each include two donor fluorescent dyeelements, designated “D002”, and one acceptor fluorescent dye element,designated “D005”.

Another example of this type of structure is the following:

Other exemplary core structures usefully incorporated into the protectedfluorescent reagent compounds of the invention include the following:

where R″ is as defined above, and “Dye” can be independently any donoror acceptor fluorescent dye element.

In some embodiments, the instant protected fluorescent compounds mayinclude a non-fluorescent multivalent central core element withattachment sites for 5, 6, or even more fluorescent dye elements on eachcore. The dyes may either be the same or different, for example, if FRETdonors and acceptors are used. Intermediate compounds containing thecore elements with attached fluorescent dyes may be further reacted withappropriate reagents to attach shield elements and binding elements asdesired, for example, as illustrated in the reaction schemes outlinedbelow.

Exemplary intermediate compounds useful in the synthesis of thejust-described embodiments include the following non-limiting examples,which comprise acetylene groups for coupling to the shield elements andtheir associated binding elements through “click” reactions,“copper-free click” reactions, or the like:

It may be advantageous under some circumstances, for example when usingthe Cu-free click reaction to assemble the protected reagent compounds,to include an azide group within the central core intermediate compound,rather than an alkyne group. Non-limiting examples of azido-containingcentral core intermediate compounds are illustrated in FIG. 12 .

After reaction of the above intermediates with a cycloalkyne-containingreagent using Cu-free click chemistry, these structures include thefollowing exemplary linkages, where the “R” group corresponds to anS′—B′ group:

Variation in the above linkages, for example where the lengths of thealkyl linker groups are altered, or where heteroatoms or otherintervening chemical moieties are substituted for the structures shown,are envisioned where such substitution does not interfere with thefunction of the linker group, as would be understood by those ofordinary skill in the art.

As noted above, the substituents labeled “Dye” and “Dye” in thesestructures may be the same or different fluorescent dye elements,depending on the desired spectroscopic properties of the respectivecompounds.

In preferred embodiments, the non-fluorescent multivalent central coreelement of these compounds is a trivalent, tetravalent, pentavalent,hexavalent, octavalent, decavalent, or dodecavalent central coreelement.

Shield Elements

As previously noted, photodamage caused by the instantly-disclosedcompounds is mitigated by the covalent incorporation of a shield elementbetween the associated fluorescent dyes and the associated bindingelements. The exact structure of the shield elements of the protectedcompounds disclosed herein are not believed to be critical, so long asthe structures are large enough to limit contacts between thefluorescent dyes and proteins, or other molecules of interest, that bindto the instant compounds and that are sensitive to photodamage by theexcited dyes. In some embodiments, the shield element comprises aprotein. In some embodiments, the shield element does not comprise aprotein.

In some embodiments, the shield element of the instant compoundspreferably comprises a shield core element that provides multivalentattachment sites for shield element side chains, where the shieldelement side chains provide the primary “bulkiness” of the shieldelement moiety and are thus believed to be responsible for theprotective effects in the compounds.

Accordingly, the shield elements may in some embodiments comprise asuitable core structure that provides for the attachment of a pluralityof side chains to the shield element core. In specific embodiments, theshield element comprises the structure:

wherein each y is independently an integer from 1 to 6. In otherspecific embodiments, the shield element comprises one of the otherbranching elements described in more detail above.

In some embodiments, the shield core elements provide a “layered”multivalent structure, such that one type of side chain can be attachedto the portion of the shield element facing the interior of theprotected compound (i.e., the dye region) and a different side chain canbe attached to the portion of the shield element facing the exterior ofthe protected compound (i.e., the binding element region). The innerlayer is ideally designed to create a protective microenvironment forthe fluorescent dyes and thus to improve their photophysical properties(e.g., their brightness) and their photochemical stabilities. The innerlayer preferably comprises pairs of neutral or negatively chargedgroups. The outer layer defines the interactions with the solvent andthe binding partner. For uses of the protected fluorescent compounds inSMRT™ sequencing reaction systems, the outer layer is preferablydesigned to improve solubility of the compound, to improve sequencingincorporation kinetics, and to minimize undesirable interactions withthe surface of the sequencing apparatus and the enzyme. The outer layerpreferably comprises pairs of negatively charged groups, although thesegroups may be altered as desired, depending on the intended use of theprotected fluorescent compound.

Exemplary shield elements usefully incorporated into the protectedfluorescent compounds of the instant disclosure include the followingnon-limiting structures:

where R₁ and R₂ represent “inner” and “outer” side chains, respectively,the “B” group is preferably a “binding element”, and the remainingportion of the structure represents the shield core element, including,in some embodiments, a “linker” group, L, that bridges the shield layersin some of the shield element embodiments. This “linker” is preferably ashort alkyl or cycloalkyl group, such as, for example, a hexyl orcyclohexyl linker group, but other linker moieties may be suitablyemployed for this purpose. For example, L may be an alkyl, heteroalkyl,cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl linker. The shieldelement is typically attached to a multivalent central core element orto a multivalent dye element through the terminal alkyl group, typicallythrough a triazole structure.

It should be understood that the S′—B′ groups are preferablysynthetically attached to central core elements using “click” reactions,or “copper-free click” reactions, as is described in further detailbelow. The S′—B′ groups are therefore preferably labeled with an azidegroup that reacts with an acetylene group of the multivalent centralcore element or dye. It should also be understood, however, that othermethods of attachment may be used to generate protected compounds withinthe scope of the instant invention, as would be understood by those ofordinary skill in the art.

Other shield elements may include additional multivalent “branched”cores to increase the number of side chains, as shown in the followingexemplary structures:

where the R₁, R₂, and B′ groups have the meanings described in theprevious paragraph, “Y” represents a suitable trivalent group, forexample one of the trivalent groups described above in the context ofthe core element components, and the shield element is attached to thefluorescent central core element at the terminal alkyl chain, typicallythrough a triazole structure. In preferred embodiments, Y is

Some shield element structures may include three, four, or even more“layers” of side chains, for example as shown in the following formulae:-Aba-Sh(R₁)₂-Sh(R₂)₂-Sh(R₃)₂—B′; and-Aba-Sh(R₁)₂-Sh(R₂)₂-Sh(R₃)₂—Sh(R₄)₂—B′;where “Aba” is

“Sh” is a shield core element, such as, for example,

“R₁”, “R₂”, “R₃”, and “R₄” are side chains, and “B′” is preferably abinding element. It should be understood that the “R₁”, “R₂”, “R₃”, and“R₄” side chain groups may be the same or different side chains, in anycombination, as desired to achieve protection from photodamage by thereagent compounds. The shield element is attached to a multivalentcentral core element or dye through the Aba group in these examples.

An exemplary cyclooctyne-labeled triple-layer shield reagent useful inthe synthesis of a protected fluorescent reagent compound according tothis aspect of the invention is:

(“MFCO-Sb2(SG1)2-Sb2(PEG7-SG1)2-Sb2(SG1)2-14C-dC6P”), where the SG1 andPEG7 components have the structures defined below.

As is true of the shield elements generally, the exact structures of theside chain components of the shield elements are not believed to becritical, so long as they are large enough to limit contacts between thefluorescent dyes and the target biomolecules that are associated withthe binding elements of the instant protected compounds. In someembodiments, the side chain components provide a suitablemicroenvironment to improve the photophysical properties and/orphotochemical stabilities of the attached dyes. Again, the exactstructures of the side chain components are not necessarily critical, solong as they provide the desired microenvironment for the dyes.

In some embodiments, the side chains comprise polyethylene glycol. Inpreferred embodiments, the polyethylene glycol side chains comprisepolyethylene glycol with from 3 to 20 repeating ethylene oxide units. Inmore preferred embodiments, the polyethylene glycol side chains comprisepolyethylene glycol with from 4 to 10 repeating ethylene oxide units. Insome embodiments, the side chains comprise a negatively-chargedcomponent, such as, for example, a component comprising a sulfonic acid.In some embodiments, the side chains comprise a combination ofpolyethylene glycol and another component, such as, for example anegatively-charged component. In preferred embodiments, the inner layerof the shield element comprises a side chain comprising polyethyleneglycol, and the outer layer of the shield element comprises a side chaincomprising a negatively-charged component.

The side chains may additionally comprise a core structure that providesfor branching within the side chains. In some embodiments, the sidechain comprises a substituted phenyl group. In specific embodiments, theside chain comprises the structure:

wherein each x is independently an integer from 1 to 6. In more specificembodiments, each x is independently an integer from 1 to 4. In someembodiments, the side chain comprises one of the other branchingelements described in more detail below.

The side chain may, in some embodiments, comprise a dendrimer. Adendrimer (or “dendron”) is a repetitively branched molecule that istypically symmetric around the core and that may adopt a sphericalthree-dimensional morphology. See, e.g., Astruc et al. (2010) Chem. Rev.110:1857. Incorporation of such structures into the shield elements ofthe instant compounds provides for a protective effect through thesteric inhibition of contacts between the fluorescent dye element orelements and one or more biomolecules associated with the bindingelement or elements. Refinement of the chemical and physical propertiesof the dendrimer through variation in primary structure of the molecule,including potential functionalization of the dendrimer surface, allowsthe protective effects to be adjusted as desired. Dendrimers may besynthesized by a variety of techniques using a wide range of materialsand branching reactions, as is well-known in the art. Such syntheticvariation allows the properties of the dendrimer to be customized asnecessary.

In some embodiments, at least one side chain comprises a peptide chain.

In some embodiments, at least one side chains comprises apolysaccharide.

Non-limiting side chain examples include the following structures:

(corresponding to PEG7) and polyethylene glycols with other numbers ofrepeating unit;

Some side chain embodiments may include combinations of any of the abovecomponents, such as, for example, the following combination of apolyethylene and a negatively-charged side chain:

Exemplary shield element-binding element (S′—B′) reagents include thenon-limiting examples illustrated in FIG. 13 :

where the azide group represents the site of attachment to themultivalent central core element or multivalent dye, typically through a“click” reaction, or a “copper-free click” reaction, and “B′” ispreferably a binding element.

In some embodiments, the molecular weight of the side chain is at least300, 350, 400, 450, or even higher. In preferred embodiments, themolecular weight of the side chain is at least 300.

Binding Elements

The protected fluorescent reagent compounds of the instant disclosurefurther comprise at least one binding element. As already described, thebinding element is responsible for recognition of a compound by a targetbiomolecule of interest, for example, an enzyme, such as DNA polymerase,when the fluorescent compound serves as a reagent in an enzymaticreaction. In some cases, the target biomolecule of interest may be areceptor, antibody, nucleic acid sequence, or the like, and the bindingelement will accordingly be selected to be specifically and efficientlyrecognized by that particular target molecule, as would be understood bythose of ordinary skill in the art.

In the case of fluorescent reagent compounds for use in single-moleculereal-time nucleic acid sequencing reactions, the binding element of theinstant protected compound comprises a nucleotide. The nucleotideportion of the protected fluorescent compound is preferably attached tothe shield element through a polyphosphate moiety coupled to thenucleotide at the normal 5′ position. With this attachment, when thenucleotide monophosphate portion of the nucleotide analog isincorporated into the growing nucleic acid strand by the DNA polymerase,the portion of the nucleotide analog containing the shield element(s)and the fluorescent dye element(s) is cleaved from the nucleotide thatis incorporated into the polynucleotide, and it diffuses away to allowfor incorporation of the next nucleotide into the chain withoutinterference from these moieties. In addition, due to the multivalencyof binding elements in the instant protected compounds, the cleavedcompounds can continue to be processed by the polymerase enzyme, so longas the compounds have remaining attached binding elements.

In preferred embodiments, the polyphosphate moiety of the instantcompounds is coupled to the shield element through a linker moiety. Thelinkers are typically short alkyl, or cycloalkyl, moieties, in somecases with heteroatom substitutions, as would be understood by those ofordinary skill in the art.

As noted above, not all B′ terminal chemical groups need be bindingelements, and different binding elements may be present in a singleprotected reagent compound.

Exemplary binding elements of the instant protected reagent compounds,in particular binding elements comprising nucleotides, are described indetail above.

In some embodiments of the instant protected reagent compounds, thebinding element is biotin. Suitable target biomolecules for compoundscomprising biotin include, for example, avidin, streptavidin, and thelike.

In some embodiments, the binding element is a nucleic acid or modifiednucleic acid. Suitable target biomolecules for compounds comprising suchbinding elements include, for example, complementary nucleic acids.

Synthesis of the Protected Fluorescent Reagent Compounds

The protected compounds of the instant disclosure are synthesized usingstandard chemical techniques. For example, exemplary shield coreelements of the instant compounds may be synthesized according to thereactions illustrated in Scheme 1 of FIG. 14 .

Multivalent fluorescent dye core elements of the instant compounds maybe synthesized, for example, according to the reactions illustrated inScheme 2 of FIG. 15 .

It should be understood that Scheme 2 can be readily adapted to allowsynthesis of variant compounds for use in FRET analyses by substitutionof one or more of the illustrated fluorophore moieties for anappropriate FRET donor or acceptor fluorophore, as would be understoodby those of ordinary skill in the art.

Non-fluorescent multivalent central core elements of the instantcompounds may be synthesized, for example, according to the reactionsillustrated in Scheme 3:

Intermediate fluorescent core elements comprising FRET donor andacceptor fluorescent dyes may be synthesized, for example, according tothe reactions illustrated in Scheme 4 of FIG. 19 .

Branching of the fluorescent core elements may be increased, forexample, as illustrated in Scheme 5 of FIG. 20 .

Alternative core elements comprising fluorescent dye elements,optionally FRET donor and acceptor fluorescent dye elements, may besynthesized, for example, according to the reactions illustrated inSchemes 6-1 to 6-4 of FIG. 16 . wherein Core 3 and Core 6 have thestructures shown in FIG. 17 .

Another example of a core element comprising fluorescent dye elements isshown in FIG. 18 , where the dyes themselves are bivalent.

The final fluorescent protected reagent compound products may begenerated by reaction of the fluorescently-labeled multivalent centralcore elements with azide-substituted shield element-binding element(“S′—B”) reagents, for example as shown in Scheme 9 of FIG. 21 .

Shield elements modified with a nucleotide hexaphosphate may besynthesized, for example according to Schemes 7-1 or 7-2 (see alsoExample 5):

The shield core element reagent, TFA-Sh-CONHS, used in the initial stepof the first two reaction cycles of Scheme 7-1, may be generated byreaction of the “Sh” shield core element of Scheme 1 with TFA-NHS toform the following structure:

SG1-N₃ has the structure:

PEG7-N3 has the structure:

N3-Aba-CONHS has the structure:

NH₂-14C-dN6P represents a hexaphosphate deoxynucleotide containing a14-carbon, or equivalent, linker chain terminating in an amino group. Anexemplary species of this structure is:

wherein the nucleobase is thymine, and the C14-linker chain includes anamide bond.

Alternative pathways for generating shield element-binding elementreagents useful in the synthesis of protected reagent compounds of theinstant disclosure are outlined in Schemes 7-3 to 7-5:

Exemplary synthetic reactions useful in the generation of theazide-containing sidechain reagents of Schemes 7-1 to 7-5 (e.g., R₁—N₃and R₂—N₃ of Schemes 7-3 to 7-5) are outlined in Scheme 8:

Reasonable variations in all of the above shield component structuresshould be considered within the scope of the invention.

Exemplary protected compounds of the instant disclosure may beassembled, for example, by the reactions illustrated in Scheme 9 of FIG.21 , wherein the azide-substituted shield element-binding element(“S′—B′”) reagent may be abbreviated as follows:

Alternatively, the core elements of the instant compounds may besynthesized, for example, by the reactions exemplified in Scheme 10:

The three products generated according to Scheme 10 illustrate the greatflexibility of the instant synthetic methods. In particular, theleft-most product contains 6 donor fluorophores attached to themultivalent central core unit, whereas the two right-most productscontain 4 donor fluorophores and 1 acceptor fluorophore attached to themultivalent central core unit. The right-most products differ in thespacing between the acceptor fluorophore and the central core element.The synthetic approaches thus provide for the synthesis of a widevariety of protected fluorescent reagents for use in FRET analyses. Thefinal products may be generated by reaction of the fluorescently-labeledmultivalent central core elements with a sufficient excess ofazide-substituted shield element-binding element (“S′—B′”) reagent, forexample as shown in Scheme 9 of FIG. 21 .

Similar approaches have resulted in the exemplary protected fluorescentreagent compounds of FIGS. 22A-22H, which include adenine and cytosinenucleobases, respectively.

Exemplary synthetic approaches useful in the preparation of dye corescontaining 6 and 9 azide terminal groups are shown in Schemes 11 and 12:

Such cores may be reacted with alkyne-containing groups, for exampleusing click chemistry or copper-free click chemistry, to generate usefulprotected fluorescent reagent compounds.

Exemplary synthetic schemes to generate the azide scaffolds employed inthe reactions of Schemes 11 and 12 may be prepared as illustrated inSchemes 13 and 14:

An exemplary synthetic approach for preparing an alkyne-substitutedshield element-binding element (S′—B′) usefully reacted with, forexample, the above-described azide-substituted dye cores is shown inSchemes 15-1 and 15-2, where the R₁ and R₂ side chain groups arePEG7-SG1 and SG1, respectively, and the binding elements are nucleotidebinding elements:

It should be understood that the shield elements exemplified in Schemes15-1 and 15-2 correspond to “two-layer” shield elements, but thatalternative shield element structures, such as three-layer, four-layer,and so on, may be readily synthesized by routine extension of thedisclosed reaction scheme. For example, the triple-layer shield reagentdescribed above could be synthesized by activating the carboxylate groupof the two-layer intermediate and reacting with an additional “Sb2”reagent. The R₁ and R₂ side chain groups, and the side chain groups ofany additional layers, may be chosen to provide the desired shieldingeffects.

Exemplary reactions for preparing components of the just-describedshield elements are illustrated in Schemes 16-1 and 16-2:

An exemplary reaction showing the attachment of alkyne-substitutedshield element-binding elements (S′—B′s), to an azide-substituted dyecore is illustrated in Scheme 17 of FIG. 23 .

As would be understood by those of skill in the art, the dye corereactant in this reaction contains 9 azide groups, and 9 molecularequivalents of the alkyne-substituted shield element-binding elementreactant would therefore be provided in order for the reaction to go tocompletion. It would also be understood that the binding element in theshield element-binding element reactant of this reaction is a nucleotidebinding element. The product of the reaction of Scheme 17 in FIG. 23 isshown as a cartoon to illustrate the expected relationship between thecentral dye core, the shield layers, and the nucleotide bindingelements, but no specific three-dimensional structure should be assumedfor the product of this reaction or any of the other protectedfluorescent reagent compounds of the disclosure.

The above examples provide a representative sample of the structuralvariety available using the synthetic approaches described herein. Inparticular, these examples illustrate variation in the multivalentcentral core element, including fluorescent and non-fluorescentmultivalent central core elements, in the fluorescent dye elements, inthe branching elements, in the shield core elements, in the shield sidechains, and in the binding elements, including linkers and nucleobases.Also illustrated are variation in placement of the reactive groups inthe coupling reactions, e.g., the azide group and the alkyne group forclick chemistry reactions, on the different precursor components of theprotected compounds. Other variants are well within the skill of theordinary artisan in view of the instant disclosure, in particular inview of the instant synthetic schemes.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein may be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following Examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES Example 1. Synthesis of Protected Fluorescent Reagent CompoundsComprising Multivalent Fluorescent Dye Elements

Three different protected fluorescent reagent compounds comprisingtetravalent fluorescent core elements were synthesized according to thefollowing experimental procedures, as outlined in Schemes 18-20 of FIGS.24-26 .

Tetracarboxy Carbocyanine Dye (D010)

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate (85.4mg, 231 umol, prepared from 1-hydrazinylbenzene-1,3-dicarboxylic acid in2 steps following the standard Fischer indole synthesis procedures) andortho-triethylformate (300 μL) in pyridine was heated at 100° C.overnight under nitrogen. Solvent was evaporated off under reducedpressure to give a dark red residue, which was then purified byreverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) to give 122 μmoleof the product (53% yield). λmax (545 nm).

Tetraalkyne Carbocyanine Dye (D010-(PA)₄)

To a solution of the tetracarboxy dye, D010 (10 μmol) in DMF (300 μL)was added CDI (60 μmop and NHS (60 μmop and stirred overnight undernitrogen. To the solution was added diethyl ether (3 mL). The resultingprecipitate was collected, washed with diethyl ether (3×1 mL) and driedunder high vacuum to give the activated NHS ester. To a solution of theactivated NHS ester (4 μmop in DMF (200 μL) was added DIPEA (20 μL)followed by addition of large excess of propargyl amine (26 μL, 400μmol). The resultant solution was stirred overnight and the crudeproduct was subjected to reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) purification to give 1.6 umol of the desired product togetherwith some incomplete reaction adducts (trialkynes and dialkynes).

D010-[Aba-Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P]₄

To a solution of D010-(PA)₄ (0.1 μmol) andN3-Aba-Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P (1.0 μmop in 2 M TEAA aqueous buffer(20 μL) was added sodium ascorbate (2.5 μL, 200 mM), copper sulfate (2μL, 100 mM), copper chelator (200 mM, 1 μL) in a vial. After vortexingfor 30 seconds the vial was placed in the dark at room temperatureovernight. To the vial was added 1M EDTA (10 μL) and the solution wassubjected to ion-exchange HPLC (0.05 M TEAA with 20% CH₃CN/1.5 M TEAAwith 20% CH₃CN) followed by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) purification to give 0.054 μmol of the desired product.

Octaalkyne Carbocyanine Dye, D010-(Sh)₄

To a solution of the activated NHS ester of the tetracarboxy dye,D010-(NHS)₄ (1 μmol), in DMF (100 μL) was added DIPEA (5 μL) followed byaddition of Sb1 (10 μmol, from Scheme 1) in DMF (50 μL). The resultantsolution was stirred overnight and the crude product was subjected toreverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) purification togive 0.56 μmol of the desired product together with some incompletereaction adducts.

D010-Sh{[Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P]₂}₄

To a solution of D010-(Sh)₄ (0.06 μmol),N₃-Aba-Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P (1.0 μmol) in 2 M TEAA aqueous buffer(20 μL) was added sodium ascorbate (2.5 μL, 200 mM), copper sulfate (2μL, 100 mM), copper chelator (200 mM, 1 μL) in a vial. After vortexingfor 30 seconds the vial was placed in the dark at room temperatureovernight. To the vial was added 1M EDTA (10 μL) and the solution wassubjected to ion-exchange HPLC (0.05 M TEAA with 20% CH₃CN/1.5 M TEAAwith 20% CH₃CN) followed by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) purification to give 0.036 μmol of the desired product.

Trialkyne Sh Compound, Sh-PA

To a solution of TFA-Sh-CONHS (14.1 mg, 29.3 μmol) in DMF (1 mL) wasadded excess amount of propargyl amine (94 μL, 1.47 mmol) and stirred atroom temperature for 2 hours. The crude product was subjected toreverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) purification togive 12.0 mg (quantitative yield) of the TFA protected product. To theTFA protected Sh-PA in acetonitrile (500 μL) was added NH₄OH (50%, 500μL) and stirred overnight. The crude product was purified byreverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) separation to give5.9 mg (62%) of the desired product.

Dodecaalkyne Carbocyanine Dye, D010-(Sh-PA)₄

To a solution of the activated NHS ester of the tetracarboxy dye,D010-(NHS)₄ (2.2 μmol), in DMF (165 μL) was added DIPEA (50 μL) followedby addition of Sh-PA (5.9 mg, 18.2 μmol) in DMF (400 μL). The resultantsolution was stirred overnight and the crude product was subjected toreverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) purification togive 0.33 μmol of the desired product together with some incompletereaction adducts.

D010-Sh{[Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P]₂}₄

To a solution of D010-(Sh-PA)₄ (0.06 μmol) andN3-Aba-Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P (1.0 μmol) in 2 M TEAA aqueous buffer(20 μL) was added sodium ascorbate (2.5 μL, 200 mM), copper sulfate (2μL, 100 mM), copper chelator (200 mM, 1 μL) in a vial. After vortexingfor 30 seconds the vial was placed in the dark at room temperatureovernight. To the vial was added 1M EDTA (10 μL) and the solution wassubjected to ion-exchange HPLC (0.05 M TEAA with 20% CH₃CN/1.5 M TEAAwith 20% CH₃CN) followed by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) purification to give 0.075 μmol of the desired product.

Example 2. Synthesis of Protected Fluorescent Reagent CompoundsComprising Non-Fluorescent Multivalent Core Elements

Protected fluorescent reagent compounds comprising non-fluorescentmultivalent core elements were synthesized according to the followingexperimental procedures. In particular, the non-fluorescent central corewas synthesized as outlined in Scheme 21, the dye reagents were preparedas outlined in Scheme 22, and the dyes were attached to the central coreas outlined in Schemes 23 and 24.

3,3′,3″-(benzene-1,3,5-triyltris(oxy))tris(propan-1-amine) (2-1b)

NaH (108 mg, 4.5 mmol) was added to Phloroglucinol (126 mg, 1 mmol) DMFsolution slowly, then tert-butyl (3-chloropropyl)carbamate (935 mg, 4.5mmol) was added and heated to 55° C. overnight. The resultant solutionwas diluted in 30 mL ethyl acetate, washed with 20 mL water 4 times.Organic layer was collected, after evaporating the solvent, the residuewas further purified with silica gel column to afford 146 mg product2-1a (24% yield) as a white solid. A mixture of 100 μL TFA and 100 μLdichloromethane was added to 2-1a to remove Boc protection group. Allsolvents are evaporated off after incubating at room temperature for 2hr. The triamino derivative 2-1b was directly used without furtherpurification.

N-(5-aminopentyl)-3,4,5-tris(hex-5-yn-1-yloxy)benzamide (2-2b)

3,4,5-tris(hex-5-yn-1-yloxy)benzoic acid (50 mg, 0.12 mmol), EDC.HCl (40mg, 0.21 mmol), N-Hydroxysuccinimide (25 mg, 0.22 mmol), 72 μL ofN,N-Diisopropylethylamine (0.4 mmol) and N-Boc-cadaverine (40 mg, 0.20mmol) were mixed in 500 μL dichloromethane and stirred overnight, theresultant mixture was purified by silica gel column using ethyl acetateand hexane as eluent to give 26 mg white solid 2-2a (44% yield). Bocprotection group was removed by incubating in the mixture of 50 μL TFAand 50 μl dichloromethane for 1 hr. Solvent was evaporated off underhigh vacuum to give a light yellow residue 2-2b which was used withoutfurther purification.

Trihexnyl gallate-5C-Asp (2-2c)

N-(5-aminopentyl)-3,4,5-tris(hex-5-yn-1-yloxy)benzamide (2-2b, 25 mg, 50μmol), Boc-asp(otbu)-osu (39 mg, 0.1 mmol) were dissolved in 0.5 mLdichloromethane, then 100 μl N,N-Diisopropylethylamine was added andstirred at RT overnight. The reaction mixture was purified by silica gelcolumn to afford 30.4 mg which solids (80% yield). After treatment with50% TFA in dicholormethane at room temperature for 2 hr, 35 μmol aminoacid 2-2c was obtained as a white solid after reverse-phase HPLCpurification.

Trihexnyl gallate-5C-Asp-D005 (2-2d)

5 μmol of D005-OSu, 5 μmol Trihexnyl gallate-5C-Asp and 10 μl DIEA weredissolved in 100 μL DMA and vortexed at room temperature for 2 hr, thereaction mixture was subjected into RP-HPLC to afford 4.5 μmol product2-2d (90% yield). The same procedure was applied to make Trihexnylgallate-5C-Asp-X-D002 2-2e. (See Scheme 22.)

Synthesis of [(D002X)₂, D005]-TS6 (2-4b) Trihexnylgallate-5C-Asp-D005-triamine (2-4a)

1 μmol of Trihexnyl gallate-5C-Asp-D005 (2-2d) was dissolved in 100 μlDMA, 5 μmol of NHS, 5 μL DIEA and 5 μmol of TSTU were addedsequentially, after vortexing at RT for 1 hr, to the solution was addedethyl acetate (2 mL). The resulting precipitate was collected, washedwith ethyl acetate (3×1 mL) and dried under high vacuum to give theactivated NHS ester. To a solution of the activated NHS ester (1 μmop inDMF (50 μL) was added excess of3,3′,3″-(benzene-1,3,5-triyltris(oxy))tris(propan-1-amine) (2-1b, 50 μL,3 μmop in 50 μL 0.2N NaHCO₃. The resultant solution was stirred for 1 hrand the crude product was subjected to reverse-phase HPLC(acetonitrile/0.1 M TEAB gradient) purification to give 0.7 μmol of thedesired product 2-4a (70% yield).

[(D002X)₂, D005]-TS6 (2-4b)

2.5 μmol of Trihexnyl gallate-5C-Asp-X-D002 was dissolved in 100 μl DMA,10 μmol of NHS, 10 μL DIEA and 10 μmol of TSTU were added sequentiallyafter vortexing at RT for 1 hr, to the solution was added ethyl acetate(2 mL). The resulting precipitate was collected, washed with ethylacetate (3×1 mL) and dried under high vacuum to give the activated NHSester. To a solution of the activated NHS ester (2.5 μmop in DMF (50 μL)was added Trihexnyl gallate-5C-Asp-D005-triamine (2-4a, 50 μL, 0.7 μmol)in 20 μL 0.2N NaHCO₃. The resultant solution was stirred for 4 hr andthe crude product was subjected to reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) purification to give 0.4 μmol of the desired product2-4b (57% yield). (See Scheme 24.)

[(D002X)₂, D005]-TS3 (2-3b)

This compound was synthesized following the same protocol by usingtriazine instead of3,3′,3″-(benzene-1,3,5-triyltris(oxy))tris(propan-1-amine) 2-1b. (SeeScheme 23 of FIG. 27 .)

The final compounds were assembled by reacting compounds 2-3b and 2-4bwith appropriate azide-substituted shield element-binding element(S′—B′) reagents using standard click conditions, as shown in Schemes 23and 24 of FIG. 27 .

Example 3. Synthesis of Alternative Protected Fluorescent ReagentCompounds Comprising Non-Fluorescent Multivalent Core Elements

Alternative protected fluorescent reagent compounds comprisingnon-fluorescent multivalent core elements were synthesized according tothe following experimental procedures. In particular, thenon-fluorescent core was synthesized as outlined in Scheme 25. The dyereagents were attached to the non-fluorescent core as described in theexperimental methods, and the final products were assembled using clickreactions as outlined in Scheme 26 of FIG. 28 .

4-(3-Aminopropyl)-N-[3,5-bis({[4-(3-aminopropyl)-3,5-bis(hex-5-yn-1-yloxy)benzene]amido})cyclohexyl]-3,5-bis(hex-5-yn-1-yloxy)benzamide,(NH₂—Sb2)₃-Chx, “CS2 core”, 3-4)

BOP (35.4 mg, 80 μmol) was added to a vial containing4-(3-{[(tert-butoxy)carbonyl]amino}propyl)-3,5-bis(hex-5-yn-1-yloxy)benzoicacid (3-1), (77.8 mg, 165 μmop, cyclohexane-1,3,5-triamine (3-2), (5.2mg, 40 μmol) and DIEA (28 μL). After stirring for 45 min at roomtemperature the reaction was partitioned between water and ethylacetate. The organic layer was separated and the aqueous layer extractedwith ethyl acetate (2×). The organic layers were combined, washed withsat. aq. NaHCO₃, brine, dried over Na₂SO₄, filtered and concentrated toa yellow oil that was subjected to normal phase purification (12 gsilica gel, 0-100% Hex:EtOAc, Combiflash) to give tert-butyl3,3′,3″-(4,4′,4″-(cyclohexane-1,3,5-triyltris(azanediyl))tris(oxomethylene)tris(2,6-bis(hex-5-ynyloxy)benzene-4,1-diyl))tris(propane-3,1-diyl)tricarbamate(3-3), (Boc-Sb2)3-Chx. The resulting white residue was dissolved in 30%trifluoroacetic acid in dichloromethane (5 mL) and allowed to stir for 1h at room temperature. The reaction was then concentrated to give4-(3-aminopropyl)-N-[3,5-bis({[4-(3-aminopropyl)-3,5-bis(hex-5-yn-1-yloxy)benzene]amido})cyclohexyl]-3,5-bis(hex-5-yn-1-yloxy)benzamide,(NH₂—Sb2)3-Chx, “CS2 core” (3-4) (20.1 mg, 16.9 μmol, 42% yield). LCMS:Calculated Mass 1188.72, Observed Mass 1188.45 (M⁻).

D005-CS2-(NH₂)₂ (3-5)

DIEA (6.3 μl, 36 μmol, 9 eq) was added to a solution of CS2 core (9.2mg, 6 μmol, 1.5 eq) and the acceptor dye D005-NHS ester (4 μmol, 1 eq)in DMA (200 μl). The mixture was vortexed at room temperature in darkfor 24 h. The product was purified by a reverse phase HPLC (WatersXTerra C18 RP 30×100, 20-54% AcN in 0.1 M TEAB, Akta Purifier) to givecompound 3-1 (4.9 mg, 1.9 μmol, 48% yield). LCMS: Calculated Mass2262.83, Observed Mass 1131.3 (M²⁻/2).

[(D002)₂, D005]-CS2 (3-6)

DIEA (4.0 μl, 23 μmol, 12 eq) was added to a solution of D005-CS2-(NH₂)₂(3-5) (4.9 mg, 1.9 μmol, 1 eq) and the donor dye D002 NHS ester (7 μmol,3.7 eq) in DMF (350 μl). The mixture was vortexed at room temperature indark for 26 h. The product was purified by a reverse phase HPLC (WatersXTerra C18 RP 30×100, 0-42% AcN in 0.1 M TEAB, Akta Purifier) to givecompound 3-6 (0.88 μmol, 46% yield). LCMS: Calculated Mass 4283.08,Observed Mass 1427.55 (M³⁻/3).

[(D002)₂, D008]-CS2-[Aba-Sh(PEG7-SG1)₂-Sh(SG1)₂-14C-dA6P]₆ (3-8)

Aqueous solutions of an alternative dye core [(D002)₂, D008]-CS2 (3-7)(50 nmol, 1 eq) and the azido shield element-binding element(N₃-Aba-Sh(PEG7-SG1)₂-Sh(SG1)₂-dA6P, 1000 nmol, 20 eq) were lyophilizedand dissolved in a mixture of water (15 μl), aqueous TEAA (2 M, 2 μl),and DMA (11 μl). In a separate vial, an aq. solution of sodium ascorbate(1 M, 2.5 μl, 2.5 μmol, 50 eq) was added to a mixture of copper(II)sulfate (100 mM, 1.0 μl, 100 nmol, 2 eq) and two copper chelators (100mM, 1.0 μl, 100 nmol, 2 eq) and (200 mM, 0.5 μl, 100 nmol, 2 eq) inwater. The copper complex solution was added to the solution of bothstarting materials and the mixture was vortexed at room temperature indark for 4.7 h. The product was purified by ion exchange chromatographyon Q HP Sepharose (GE, 5 ml column, 0.05-1.5 M TEAB in 30% AcN, AktaPurifier) followed by a reverse phase HPLC (Waters XTerra C18 RP 19×100,0-27% AcN in 0.1 M TEAB, Akta Purifier) to give compound 3-8 (24 nmol,48% yield, 323 μM, 75 μl).

Example 4. Synthesis of Exemplary Shield Core Elements

Exemplary intermediate compounds useful in the synthesis of the shieldcore elements are outlined in Scheme 27.

Methyl 3,5-dihydroxy-4-iodobenzoate (4-2)

To a vial containing methyl 3,5-dihydroxybenzoate (4-1) (5.0 g, 30 mmol)in methanol (70 mL) and NaHCO₃ (70 mL, 1 M) was added, via syringe pump(12 mL/min) at 0° C., a solution of iodine (7.4 g, 29.3 mmol) in aqueouspotassium iodide (10 mL, 3.8 M). After 1 h the reaction was quenched topH 2 with HCl (37%) and then allowed to warm to room temperature. Thereaction was concentrated to dryness and recrystallized frommethanol-water. The final product, methyl 3,5-dihydroxy-4-iodobenzoate(4-2), was isolated as yellow crystals (4.4 g, 14.8 mmol, 49% yield).LCMS: Calculated Mass 293.94, Observed Mass 293.03 (M⁻).

Methyl 3,5-bis(benzyloxy)-4-iodobenzoate (4-4)

A mixture of methyl 3,5-dihydroxy-4-iodobenzoate (4-2) (2.94 g, 10.00mmol), benzyl bromide (3.56 mL, 5.13 g, 30.00 mmol) and cesium carbonate(9.77 g, 30.00 mmol) in acetone (25 mL) was stirred at 40° C. under Arfor 4 h. The mixture was concentrated in vacuo, diluted with water (100mL) and extracted with ethyl acetate (2×150 mL). The combined organiclayers were dried over sodium sulfate, filtered and concentrated. Thecrude product was purified by column chromatography (silica gel,hexane-ethyl acetate, Combiflash) to give methyl3,5-bis(benzyloxy)-4-iodobenzoate (4-4) (2.09 g, 4.41 mmol, 44% yield)as a white solid. NMR (CDCl₃, 300 MHz): 3.92 (s, 3H); 5.22 (s, 4H); 7.23(s, 2H); 7.33-7.44 (m, 6H); 7.53-7.56 (m, 4H).

Methyl3,5-bis(benzyloxy)-4-(3-{[(tert-butoxy)carbonyl]amino}prop-1-yn-1-yl)benzoate(4-6)

To a vial containing methyl 3,5-bis(benzyloxy)-4-iodobenzoate (4-4) (1.3g, 2.4 mmol), copper(I) iodide (52.3 mg, 275 μmol), andtetrakis(triphenylphosphine)palladium(0) (158.7 mg, 137 μmol) was addeddiisopropylamine (15 mL) and dimethylformamide (15 mL). The dark brownsolution was degassed with Ar and to this was added N-Boc-propargylamine(4-5) (1.3 mg, 8.2 mmol). The reaction was allowed to stir under Ar at80° C. for 5 h. The reaction was then quenched with aqueous ammoniumsulfate (150 mL, saturated) and extracted with ethyl acetate (2×150 mL).The organic layers were combined, washed with brine, dried over sodiumsulfate, filtered and concentrated to an orange oil that was subjectedto normal phase purification (24 g silica gel, 0-25% Hex:EtOAc,Combiflash). The product was isolated as a pale orange solid which wassonicated in methanol and then filtered to give, methyl3,5-bis(benzyloxy)-4-(3-{[(tert-butoxy)carbonyl]amino}prop-1-yn-1-yl)benzoate(4-6), as an off white solid (600 mg, 1.2 mmol, 50% yield). ¹H NMR(CDCl₃, 300 MHz): 1.5 (s, 9H); 3.9 (s, 3H); 4.3 (d, 2H); 5.2 (s, 4H);5.8 (bs, 1H); 7.2-7.5 (m, 12H).

Methyl4-(3-{[(tert-butoxy)carbonyl]amino}propyl)-3,5-bis(prop-2-yn-1-yloxy)benzoate(4-9)

To a vial containing methyl3,5-bis(benzyloxy)-4-(3-{[(tert-butoxy)carbonyl]amino}prop-1-yn-1-yl)benzoate(4-6) (5.9 g, 1.2 mmol) in ethyl acetate (4.2 mL) was added palladium oncarbon (500 mg, 10% water). The reaction was degassed and allowed tostir under H₂ overnight. After stirring overnight the reaction wasfiltered over Celite to give methyl4-(3-{[(tert-butoxy)carbonyl]amino}propyl)-3,5-dihydroxybenzoate (4-7),as a clear oil. To the oil was added cesium carbonate (1.4 g, 4.3 mmol)and dimethylformamide (3 mL) followed by propargyl bromide (447.8 mg,3.0 mmol, 80%). The reaction was sealed and allowed to stir for 5 h at80° C. The reaction was then quenched with saturated aqueous ammoniumsulfate (150 mL) and extracted with ethyl acetate (2×150 mL). Theorganic layers were combined, washed with brine, dried over sodiumsulfate, filtered and concentrated to a dark yellow film that wassubjected to normal phase Combiflash purification (24 g silica gel,0-20% Hex:EtOAc, Combiflash). The isolated product methyl4-(3-{[(tert-butoxy)carbonyl]amino}propyl)-3,5-bis(prop-2-yn-1-yloxy)benzoate(4-9), appeared as a white solid (358.6 mg 893.3 mol, 68% yield). ¹H NMR(CDCl₃, 300 MHz): 1.4 (s, 9H); 1.7 (m, 2H); 2.5 (t, 2H); 2.8 (m, 2H);3.1 (m, 2H); 3.9 (s, 3H); 4.8 (d, 4H); 4.9 (bs, 1H); 7.3 (s, 2H).

4-(3-Aminopropyl)-3,5-bis(prop-2-yn-1-yloxy)benzoic acid, “Sh” (4-10)

To a solution of methyl4-(3-{[(tert-butoxy)carbonyl]amino}propyl)-3,5-bis(prop-2-yn-1-yloxy)benzoate(4-9) (357 mg, 890 mmol) in dioxane (4 mL) was added sodium hydroxide(4.4 mL, 1 M). The reaction was sealed and allowed to stir overnight at50° C. The reaction was then concentrated, acidified to pH 3.5 withcitric acid (5%) and extracted with DCM (3×50 mL). The organic layerswere combined, washed with brine, dried over magnesium sulfate, filteredand concentrated to a white solid. To the solid was added 30%trifluoroacetic acid in dichloromethane (5 mL) and the reaction wasallowed to stir at room temperature for 2 h. The reaction was thenconcentrated, redissolved in water, neutralized to pH 7 with NaHCO₃ (1M) and the resulting product was collected by filtration to give thedesired product 4-(3-aminopropyl)-3,5-bis(prop-2-yn-1-yloxy)benzoic acid(4-10), as a white solid (256 mg, 890 μmol, 100% yield). LCMS:Calculated Mass 287.12, Observed Mass 285.99 (M⁻). ¹H NMR (d6-DMSO, 300MHz): 1.7 (m, 2H); 2.6 (t, 2H); 2.7 (m, 2H); 3.6 (t, 2H); 4.9 (d, 4H);7.3 (s, 2H); 7.7 (bs, 3H).

4-(3-Aminopropyl)-3,5-bis(hex-5-ynyloxy)benzoic acid, “Sb2” (4-13)

4-(3-Aminopropyl)-3,5-bis(hex-5-ynyloxy)benzoic acid, “Sb2” (4-13) wasprepared from methyl4-(3-(tert-butoxycarbonylamino)propyl)-3,5-dihydroxybenzoate (4-7) in asimilar synthetic sequence using 6-chloro-1-hexyne instead of propargylbromide, as outlined in Scheme 28.

Example 5. Synthesis of Exemplary Binding Element-Shield ElementReagents

Exemplary reactions useful in the attachment of binding elements, forexample binding elements comprising nucleobases, to shield elements inthe generation of binding element-shield element (“S′—B”) reagents areoutlined in Scheme 29 of FIG. 29 .

TFA-Sh-14C-dT6P (5-3)

A solution of TFA-Sh-CONHS (5-1), (36.0 mg, 75 μmol) in DMA (1.1 mL) wasadded to an aq. solution of NH₂-14C-dT6P (5-2) (618 μL, 81.0 mM) andNaHCO₃ (4.1 mg, 48.8 μmop. The resulting solution was allowed to stirovernight at room temperature and the crude product was subjected toreverse phase HPLC purification (Waters XTerra C18 RP 30×100, 0-37% AcNin 0.1 M TEAB, Akta Purifier) to give intermediate reagent 5-3 (29.7μmol, 59.5 mM, 59% yield). LCMS: Calculated Mass 1299.16, Observed Mass1297.78 (M²⁻/2).

NH₂-Sh(SG1)₂-14C-dT6P (5-5)

Ammonium hydroxide (2 mL, 14.8 M) was added to a solution ofTFA-Sh-14C-dT6P (5-3) (499 μL, 59.5 mM), triethylamine (297 μL) andacetonitrile (500 4). The reaction was allowed to stir at roomtemperature for 3 days. The crude product was then evaporated to drynessand subjected to reverse phase HPLC purification (Waters XTerra C18 RP30×100, 0-37% AcN in 0.1 M TEAB, Akta Purifier). A 100 mM aq. solutionof the product was prepared and to this was added TEAA (29.7 μL, 2 M)and3-{4-[(3-azidopropyl)carbamoyl]-2,6-bis(3-sulfopropoxy)phenoxy}propane-1-sulfonicacid (5-4) (445.5 μL, 200 mM). In a separate vial, an aq. solution ofsodium ascorbate (148.5 μL, 1 M) was added to a mixture of copper(II)sulfate (14.8 μl, 1 M). The copper complex solution was added to thesolution of both starting materials and the mixture was allowed to stirat room temperature in the dark for 3 h. The crude product was purifiedby ion exchange chromatography on Q HP Sepharose (GE, 5 ml column,0.05-1.5 M TEAB in 20% AcN, Akta Purifier) followed by a reverse phaseHPLC purification (Waters XTerra C18 RP 30×100, 0-20% AcN in 0.1 M TEAB,Akta Purifier) to give compound 5-5 (9.9 μmol, 49.4 mM, 33% yield).LCMS: Calculated Mass 2439.38, Observed Mass 1219.40 (M²⁻/2).

N3-Aba-Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P (5-10)

A solution of TFA-Sh-CONHS (5-1) (4.9 mg, 10.2 mol) in DMA (287 μL) wasadded to an aq. solution of NH₂—Sh(SG1)₂-14C-dT6P (5-5) (159 μL, 49.4mM) and NaHCO₃ (6.6 mg, 78.7 μmop. The resulting solution was allowed tostir overnight at room temperature and the crude product was subjectedto reverse phase HPLC purification (Waters XTerra C18 RP 30×100, 0-20%AcN in 0.1 M TEAB, Akta Purifier). To the product, compound 5-6, wasthen added ammonium hydroxide (2 mL, 14.8 M) and the solution wasallowed to stir overnight at room temperature. The crude product wasthen evaporated to dryness and subjected to reverse phase HPLCpurification (Waters XTerra C18 RP 30×100, 0-28% AcN in 0.1 M TEAB, AktaPurifier). A 39.8 mM aq. stock solution of the product was prepared andto this was added TEAA (20 μL, 2 M) and PEG7 azide (5-7) (21.0 mg, 47.8μmop. In a separate vial, an aq. solution of sodium ascorbate (39.8 μL,1 M) was added to copper(II) sulfate (4.0 μL, 1 M). The copper complexsolution was added to the solution of both starting materials and themixture was vortexed at room temperature overnight. The crude productwas purified by ion exchange chromatography on Q HP Sepharose (GE, 5 mlcolumn, 0.05-1.5 M TEAB in 20% AcN, Akta Purifier) followed by a reversephase HPLC purification (Waters XTerra C18 RP 30×100, 0-25% AcN in 0.1 MTEAB, Akta Purifier) to give NH₂-Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P (5-8). A100 mM aq. solution of the product was prepared and to this was addedNaHCO₃ (6.6 mg, 78.7 μmop followed by a solution of2,5-dioxopyrrolidin-1-yl 4-azidobutanoate (5-9) (59 μL, 58.8 mM) in DMA(200.6 μL). The resulting solution was allowed to stir overnight at roomtemperature and the crude product was purified by ion exchangechromatography on Q HP Sepharose (GE, 5 ml column, 0.05-1.5 M TEAB in20% AcN, Akta Purifier) followed by reverse phase HPLC purification(Waters XTerra C18 RP 30×100, 0-28% AcN in 0.1 M TEAB, Akta Purifier) togive N₃-Aba-Sh(PEG7)₂-Sh(SG1)₂-14C-dT6P (5-10) (7.1 μmol, 35.4 mM, 90%yield). LCMS: Calculated Mass 3608.98, Observed Mass 1804.05 (M²⁻/2).See FIG. 30 .

Example 6. Use of Protected Fluorescent Reagent Compounds in SequencingReactions

Sequencing reactions were carried out in a zero-mode waveguide (“ZMW”)array having 3000 discrete cores. The reactions were observed using ahighly multiplexed confocal fluorescent microscope providing a targetedillumination profile, e.g., a separate spot for each core. See, e.g.,U.S. Pat. No. 7,714,303, filed May 9, 2008, which is incorporated hereinby reference in its entirety for all purposes. Fluorescent signals fromthe various ZMWs were detected using an EMCCD camera for 10 minutes, andwere subjected to pulse recognition and base calling processes. See,e.g., U.S. Pat. No. 8,182,993, filed Jun. 5, 2008, which is incorporatedherein by reference in its entirety for all purposes. The sequencing wascarried out generally as described in Eid, J. et al. (2009) Science323:133-138, and the corresponding supplemental information includedtherewith.

For each of the sequencing reactions the laser power was 1.5 μW/μm² anda camera frame rate of 100 FPS. The template was a circular vD“SMRTbell” template of about 11000 kb as described in U.S. Pat. No.8,236,499, filed Mar. 27, 2009. The polymerase enzyme immobilized in thezero mode waveguide was a mutant Φ29 polymerase as described in U.S.Pat. No. 8,257,954, filed Mar. 30, 2009. The reaction mixture had aBis-Tris Propane pH 7.5 buffer, antioxidants, 40 mM DTT, 120 mM KOAc tocontrol ionic strength; 30 mM MgOAc and 4% organic solvent additive. Themixture also contained a set of nucleotide analogs corresponding to A,G. C, and T, each present at 150-400 nM, each having a polyphosphatechain with 6 phosphates with a unique fluorescent dyes attached to theterminal phosphate. Ten minute movies of the sequencing reactions wereobtained. Data were collected on the brightness, kinetics (pulse width,the interpulse distance (IPD)), photophysical signal stability,sequencing error types, read length, and accuracy. Regular analogs(condition 1) were replaced by a protected compound for A (condition 2),and both A and C analogs (condition 3). G and T analogs remainedunchanged throughout the experiment.

The protected fluorescent reagent compounds shown in FIGS. 3A and 3Bwere synthesized according the reaction schemes described above. Thecompound of FIG. 3A (“Compound 3A”) has the structure abbreviated as[(D002)₂, D008]-CS2-[Aba-Sh(PEG7-SG1)₂-Sh(SG1)₂-14C-dA6P]₆, where D002is a FRET donor dye, and D008 is a FRET acceptor dye, CS2 is anon-fluorescent multivalent central core element synthesized asdescribed in Example 3 (compound 3-8). The shield elements are attachedto the central core element through an Aba group, the inner shieldcomprises two PEG7-SG1 groups, the outer shield comprises two SG1groups, and the binding element, adenosine, is coupled through a14-carbon equivalent and a hexaphosphate. The compound has a maximumemission fluorescence at 618 nm. The compound of FIG. 3B (“Compound 3B”)has the structure abbreviated as [(D002)₂,D007]-CS2-[Aba-Sh(PEG7-SG1)₂-Sh(SG1)₂-14C-dC6P]₆, which is the same asthe structure of FIG. 3A, except that the acceptor dye is D007, and thebinding element is cytosine. This compound has a maximum emissionfluorescence at 648 nm.

Fluorescence excitation and emission spectra for the compounds shown inFIGS. 3A and 3B are displayed below each structure. In each case, anexcitation spectrum was taken near the maximum emission, at either 619nm for Compound 3A or at 650 nm for Compound 3B. Two emission spectraare shown for each compound (at excitations of 532 nm and 549 nm forCompound 3A and at excitations of 532 nm and 547 nm for Compound 3B).The 532 nm green laser line and the 643 nm red laser line are shown ineach panel for reference.

FIG. 4 shows the relative brightness of Compounds 3A and 3B compared toa control unprotected compound containing dye D002 (see below).Cumulative ZMW distribution is plotted as a function of C/A channelratio of pkmid fluorescence intensities. Pkmid represents a peak medianbrightness, which is derived as follows. For each incorporation signalpulse, first and last frames are taken out, and the mean number ofemitted photons detected by the camera is computed. These values arepresented in the table insert for control (condition 1), Compound 3Asubstituted for A (condition 2), and both Compound 3A substituted for Aand Compound 3B substituted for C (condition 3), respectively. Compound3A is approximately 1.25 times brighter than the unprotected compound.Compound 3B is approximately 1.5 times brighter than the unprotectedcompound. According to the pkmid values, Compound 3A is ˜1.5× brighterthan the control, and Compound 3B has about the same brightness as thecontrol. The unprotected control reagent for the A nucleotide had thefollowing structure:

FIG. 5 shows the error profile of sequencing reactions using theexemplary protected compounds under the above-described conditions. Theleft panel compares insertion rates, the middle panel compares mismatchrates, and the right panel compares deletion rates. Within each panel,results are shown for condition 1 (i.e., control unprotected compounds)(left bar), for condition 2 (i.e., Compound 3A replacing A) (middlebar), and for condition 3 (i.e., Compound 3A replacing A and Compound 3Breplacing C) (right bar). As shown in the figure, insertions were lowerfor the protected compounds, while mismatches and deletions weresomewhat higher. This result confirms that the bulkiness of theprotected compounds does not significantly affect the reliability ofsequencing using these reagents. The result also demonstrates thatenclosure of the dyes inside the protective macroscaffold reducesundesirable interactions of the dyes with ZMW surface and/or the DNApolymerase.

FIGS. 6A-6D show an analysis of readlength and accuracy for theprotected compounds. FIG. 6A compares accuracy as a function ofreadlength, wherein each dot is an aligned ZMW read, for the threeconditions (top: condition 1; middle: condition 2; bottom: condition 3).FIG. 6B provides a summary of the overall sequencing results using thethree different conditions. FIG. 6C plots the probability distributionfunction (y-axis) versus accuracy (x-axis) for the three differentconditions. FIG. 6D plots the probability distribution function (y-axis)versus readlength for the three different conditions. The results shownin FIGS. 6A-6D demonstrate that accuracies are comparable for the threedifferent conditions (using 0, 1, or 2 protected fluorescent compounds).The differences in readlengths are driven primarily by variations ininterpulse distance.

Example 7. Photostability of Protected Fluorescent Reagent Compounds inSequencing Reactions

Sequencing reactions were carried out as described in Example 6, exceptthat 45 minute movies of the sequencing reactions were obtained at laserpower 2.5 μW/μm², and one additional combination of protected andunprotected fluorescent reagent compounds was tested. Data correspondingto the brightness, kinetics (pulse width, the interpulse distance(IPD)), photophysical signal stability, sequencing error types, readlength, and accuracy were collected for each reaction. Non-protectedfluorescent reagent analogs (condition 1) were replaced by Compound 3Afor A (condition 2), by Compound 3B for C (condition 3), and by bothCompound 3A and Compound 3B for A and C (condition 4). G and T analogsremained unchanged throughout the experiment.

FIG. 7 shows a comparison of the brightness of the samples under thefour tested conditions. The results indicate that Compound 3A isapproximately 15% brighter than an unprotected compound containing thesame dye, and Compound 3B has approximately the same brightness as anunprotected compound containing the same dye. Use of the protectedcompounds in sequencing reactions is therefore expected to result incomparable or lower rate of missing pulses compared to unprotectedcompounds.

FIGS. 8A and 8B show a comparison of peak height variance for thevarious dye mixtures as measured in the A channel (FIG. 8A) and in the Cchannel (FIG. 8B). In particular, the peak height variance decreases forCompound 3A in the A channel (conditions 2 and 4, FIG. 8A) and forCompound 3B in the C channel (conditions 3 and 4, FIG. 8B). A samplecontaining both Compound 3A and Compound 3B shows lower peak heightvariance in both A and C channels (condition 4, FIGS. 8A and 8B). Thesedata demonstrate an increased photophysical signal stability for theprotected compounds compared to the unprotected control compounds. Theincreased stability improves the accuracy of incorporation pulse callingthrough reduction of photophysical pulse splitting.

FIGS. 9A-9C demonstrate a decrease in the occurrence of photodamage inthe sequencing reactions when using the protective compounds. Inparticular, these results demonstrate a decrease in photodamage to DNApolymerase, a critical factor in obtaining maximal readlengths withsingle-molecule real-time sequencing methods. FIG. 9A shows a plot ofthe inverse of the photodamage component of the overall polymeraselifetime (in bases incorporated) as a function of the photons receivedby the system (normalized by the difference in kinetics (pulsewidths andIPDs) between conditions). The slope of the resulting lines representsthe overall rate of photodamage for particular condition. FIG. 9B showsa plot of the probability of polymerase survival (logarithmic scale) asa function of the photons received by the system (linear scale). Theseresults demonstrate a mitigation of photodamage in the system throughthe use of the protected fluorescent compounds and, in particular, animproved probability of survival for the DNA polymerase when using theprotected compounds. An exponential fit of overall polymerase lifetimein bases is summarized in FIG. 9C for each condition. These resultsdemonstrate that photodamage resulting from the fluorescent dye issignificantly mitigated in the protected reagent compounds.Specifically, the overall photodamage tau improvement using protectedversions of the A and C nucleotides (i.e., Compounds 3A and 3B,respectively) is approximately 7-fold.

Example 8. Decreased Photodamage in Sequencing Reactions Using ProtectedFluorescent Reagent Compounds with Increased Triple-Layer Shield Number

FIGS. 10A-10C illustrate the protective effects of increasing numbers ofa triple-layered shield element in protected reagent compounds used forsingle-molecule real-time DNA sequencing reactions. FIG. 10A shows thecommon structural features of the tested protected compounds.Specifically, the tested compounds contained a dye core comprising twodonor fluorophores (“D003”) and one acceptor fluorophore (“D006”), asshown on the left side of the figure. The six “R” groups in the dye corestructure represent the shield element-binding element component of thestructures, as shown on the right side of the figure.Cycloalkyne-substituted shield element-binding element components werecoupled to an azide-substituted dye core precursor compound using acopper-free click reaction. As shown, the compounds comprise a“triple-layer” shield element that is terminated with a deoxycytosinebinding element. The tested compounds contained either 4, 5, or 6 of theshown R groups attached to the dye core. These compounds were purifiedfrom one another following the click reaction using ion-exchangechromatography. The purified compounds were characterized by acombination of enzymatic digestion (to determine nucleotideconcentration) and absorbance (to determine dye core concentration).

FIG. 10B shows the results of sequencing reactions, performed asdescribed in Example 6, but with 100 nM of the above-described protecteddeoxycytosine reagent compounds and 150 nM of protected sequencingreagents corresponding to the other three bases. As shown, the compoundswith higher substitution of the shield elements show an increasedprobability of polymerase survival (logarithmic scale) as a function ofbases sequenced (left panel) and photons received (right panel). FIG.10C shows an exponential fit of overall polymerase lifetime for each ofthe compounds from the experiments of FIG. 10B. These resultsdemonstrate a mitigation of photodamage in the system through the use ofthe compounds with increased substitution of shield layers.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein.

While specific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined by reference to theappended claims, along with their full scope of equivalents.

What is claimed is:
 1. A method of nucleic acid sequence analysis comprising: providing an array of optically confined regions, each region comprising an immobilized nucleic acid template/polymerase primer complex and a fluorescent compound in fluid contact with the immobilized nucleic acid template/polymerase primer complex; illuminating the array of optically confined regions with an excitation wavelength; and detecting fluorescent signals emitted from each region in the array of optically confined regions; wherein the fluorescent compound is of structural formula (I): Z—[S′—B′]_(m)  (I); wherein Z is a multivalent central core element comprising a fluorescent dye element; each S′ is independently an intermediate chemical group, wherein at least one S′ comprises a shield element; each B′ is independently a terminal chemical group, wherein at least one B′ comprises a binding element comprising a nucleotide; and m is an integer from 2 to
 24. 2. The method of claim 1, wherein the shield element decreases photodamage of the fluorescent compound or of a biomolecule associated with the binding element.
 3. The method of claim 1, wherein the shield element decreases contact between the fluorescent dye element and the binding element.
 4. The method of claim 1, wherein the shield element comprises a plurality of side chains.
 5. The method of claim 4, wherein at least one side chain has a molecular weight of at least
 300. 6. The method of claim 4, wherein at least one side chain comprises a dendrimer.
 7. The method of claim 4, wherein at least one side chain comprises a polyethylene glycol.
 8. The method of claim 4, wherein at least one side chain comprises a negatively-charged component.
 9. The method of claim 4, wherein at least one side chain comprises a substituted phenyl group.
 10. The method of claim 4, wherein at least one side chain comprises a triazole.
 11. The method of claim 1, wherein the shield element comprises an inner layer and an outer layer.
 12. The method of claim 1, wherein at least one B′ comprises a binding element comprising a biotin.
 13. The method of claim 1, wherein at least one B′ comprises a binding element comprising a polyphosphate.
 14. The method of claim 1, wherein Z comprises a branching element.
 15. The method of claim 1, wherein m is an integer from 2 to
 8. 16. The method of claim 1, wherein the fluorescent dye element is a cyanine dye.
 17. The method of claim 1, wherein the fluorescent dye element is a multivalent fluorescent dye element.
 18. The method of claim 1, wherein the fluorescent compound is of structural formula (III):

wherein X is a non-fluorescent multivalent central core element; at least one D is a fluorescent dye element; at least one W is a branching element; n is an integer from 2 to 6; each p′ is independently an integer from 1 to 4; and each p″ is independently an integer from 1 to
 4. 19. The method of claim 18, wherein X comprises a polyamine, a substituted cyclohexane, a substituted 1,3,5-triazine, or a substituted benzene.
 20. The method of claim 18, comprising at least one donor fluorophore and at least one acceptor fluorophore.
 21. The method of claim 18, wherein the shield element comprises a plurality of side chains.
 22. The method of claim 21, wherein at least one side chain has a molecular weight of at least
 300. 23. The method of claim 21, wherein at least one side chain comprises a dendrimer.
 24. The method of claim 21, wherein at least one side chain comprises a polyethylene glycol.
 25. The method of claim 21, wherein at least one side chain comprises a negatively-charged component.
 26. The method of claim 21, wherein at least one side chain comprises a substituted phenyl group.
 27. The method of claim 21, wherein at least one side chain comprises a triazole.
 28. The method of claim 18, wherein the shield element comprises an inner layer and an outer layer.
 29. The method of claim 18, wherein at least one B′ comprises a binding element comprising a biotin.
 30. The method of claim 18, wherein at least one B′ comprises a binding element comprising a polyphosphate.
 31. The method of claim 18, wherein n is an integer from 2 to
 4. 32. The method of claim 18, wherein each p′ is independently an integer from 1 to
 3. 33. The method of claim 18, wherein each p″ is independently an integer from 1 to
 3. 34. The method of claim 1, wherein the shield element does not comprise a protein.
 35. The method of claim 1, wherein each region in the array of optically confined regions comprises a plurality of fluorescent compounds in fluid contact with the immobilized nucleic acid template/polymerase primer complex.
 36. The method of claim 35, wherein each region in the array of optically confined regions comprises four different fluorescent compounds, each compound comprising a different nucleotide and emitting a different fluorescent signal. 